Medicine for preventing ischemic stroke and preparation method thereof

Abstract

This invention relates to a drug for preventing ischemic stroke and its preparation method, belonging to the field of biomedical technology. It comprises 5,7,4'-trihydroxyflavone and tetramethylpyrazine in a mass ratio of (62-68):(32-38), sequentially coated with EPC-Cholesterol nanoparticles, and DSPE-PE G containing CREKA peptide and sausage pulp extract peptides. 2000 - One-time modification of complex peptides, DSPE-PEG 2000 The drug was obtained through secondary modification with TK. This invention utilizes a scientifically formulated synergistic effect of 5,7,4'-trihydroxyflavone and tetramethylpyrazine, combined with the targeting action of CREKA peptide, the protective effect of sausage pulp-extracted peptides, and DSPE-PEG. 2000 The controlled release and antioxidant effects of -TK achieve multi-level synergy of thrombus targeting, gradient controlled release, inflammation inhibition, and neuroprotection, breaking through the limitations of traditional single-component drugs and improving the effectiveness of preventing neurological damage after stroke.

Description

Technical Field

[0001] This invention belongs to the field of nanotechnology in biomedicine, specifically relating to a drug for preventing ischemic stroke and its preparation method, which is a combination of neuroprotective drug delivery system and ischemia-reperfusion injury treatment technology. Background Technology

[0002] Stroke, a common neurological disease that seriously threatens human health, is characterized by high incidence, high disability rate, and high mortality rate, and is one of the leading causes of death and disability among adults worldwide. Among the many types of stroke, ischemic stroke is the most common, and its pathogenesis stems from thrombosis blocking cerebral arteries, leading to the interruption of blood supply to local brain tissue.

[0003] In the acute phase of ischemic stroke, the interruption of blood flow caused by thrombosis can lead to irreversible necrosis in the ischemic core area within a very short time, directly damaging nerve cells. During the reperfusion phase, a series of complex pathophysiological processes follow: a massive release of reactive oxygen species (ROS) causes severe imbalance in intracellular redox balance; mitochondrial function is impaired, leading to disordered energy metabolism; and the inflammatory cascade intensifies, releasing large amounts of inflammatory factors, further aggravating inflammatory damage and expanding the extent of brain injury. These interacting pathological processes significantly increase the difficulty of preventing and treating ischemic stroke.

[0004] Despite continuous advancements in the medical field, the clinical treatment outcomes for ischemic stroke remain unsatisfactory, with many pressing issues that need to be addressed:

[0005] 1. Thrombolytic therapy: Recombinant tissue plasminogen activator (tPA), which is widely used in clinical practice, can dissolve thrombi and restore blood flow, but its treatment time window is extremely narrow. It must be used within 4.5 hours after the onset of the disease. Beyond this time window, the treatment effect is greatly reduced and the risk of bleeding increases significantly, such as serious complications like intracranial hemorrhage, which limits its clinical application.

[0006] 2. Neuroprotective agents: Neuroprotective agents aim to reduce brain damage and protect nerve cells through multiple mechanisms. However, due to the high selectivity and restriction of the blood-brain barrier (BBB), most neuroprotective agents cannot effectively penetrate the blood-brain barrier and cannot reach sufficient drug concentrations in the brain, thus failing to exert the ideal neuroprotective effect.

[0007] Given the limitations of existing treatments, finding safe and effective new methods for the prevention and treatment of ischemic stroke has become an urgent priority in the field of medical research. In recent years, with a deeper understanding of the inefficiency and serious side effects of chemical drugs in treatment, natural herbs, with their advantages of multiple components, multiple targets, and low toxicity, have become increasingly important in the fields of health maintenance and disease treatment.

[0008] Flavonoids, as important active ingredients in herbal medicines, are a well-known class of natural nutrients. Due to their powerful antioxidant capacity, anti-inflammatory, and anti-apoptotic activities, they have shown great potential in disease prevention and treatment. Apigenin (5,7,4'-trihydroxyflavone), a typical representative of flavonoids, is widely found in various herbs and plants such as celery, chamomile, and perilla. Extensive basic research has shown that apigenin possesses unique neuroprotective properties in the treatment of nervous system diseases. In studies related to ischemic stroke, apigenin has been found to effectively reduce oxidative stress-induced damage to nerve cells by scavenging excess ROS; it can also inhibit the production and release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, regulating the inflammatory response and reducing neuronal damage caused by inflammation; simultaneously, apigenin can regulate intracellular signaling pathways, inhibit apoptosis, and promote nerve cell survival and repair.

[0009] Although apigenin shows promising potential in the prevention and treatment of ischemic stroke, its clinical translation still faces numerous challenges. Apigenin suffers from poor oral bioavailability, with low absorption and bioavailability in the gastrointestinal tract after oral administration; its short plasma half-life leads to rapid metabolism and elimination in the body, making it difficult to maintain effective blood concentrations; and its high lipid solubility and poor water solubility result in extremely low distribution in brain tissue, hindering its ability to effectively cross the blood-brain barrier and reach the site of brain injury. These factors severely limit the efficacy of apigenin in the treatment of ischemic stroke. Therefore, innovative drug delivery technologies, synergistic absorption enhancers, and formulation optimization strategies are needed to address these issues and further advance the development of apigenin in the treatment of ischemic stroke. Summary of the Invention

[0010] Due to the limitations of apigenin, including low bioavailability in the gastrointestinal tract and difficulty in effectively crossing the blood-brain barrier, it is difficult to exert its excellent preventive and therapeutic effects on ischemic stroke. This invention provides a drug for the prevention of ischemic stroke and its preparation method. It utilizes a scientifically designed synergistic ratio of 5,7,4'-trihydroxyflavone and tetramethylpyrazine, combined with the targeting effect of CREKA peptide, the protective effect of sausage pulp-extracted peptides, and DSPE-PEG. 2000 -TK's controlled release and antioxidant effects achieve a multi-level synergistic effect of thrombus targeting, gradient controlled release, inflammation suppression, and neuroprotection, overcoming the limitations of traditional single-component drugs and improving the effectiveness of preventing post-stroke neurological damage. Its specific technical solution is as follows:

[0011] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; wherein the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are encapsulated in EPC-Cholesterol nanoparticles, followed by DSPE-PEG. 2000 - The complex peptide was modified once, followed by DSPE-PEG. 2000 -TK secondary modification yields the drug.

[0012] In the above-mentioned drugs, the mass ratio of 5,7,4'-trihydroxyflavone to tetramethylpyrazine is (62-68):(32-38).

[0013] In the aforementioned drug, the mass ratio of EPC-Cholesterol is EPC:Cholesterol = (58-62):(18-22).

[0014] In the above-mentioned drugs, the DSPE-PEG 2000 - In the complex peptide, the mass ratio of the components of the complex peptide is CREK A peptide: sausage pulp extract peptide = (50-60): (40-50).

[0015] The preparation method of the sausage pulp extract peptide in the above-mentioned complex peptide includes: adding sausage pulp to deionized water for homogenization to make a pulp, adjusting the pH to 1.5-2.0, adding pepsin, and enzymatically hydrolyzing at 36℃-38℃ for 2-2.5h; adjusting the pH to 8.0-8.5, adding trypsin, and enzymatically hydrolyzing at 36℃-38℃ for 1-1.5h, inactivating the enzyme, centrifuging to collect the supernatant, ultrafiltration using an ultrafiltration membrane, taking the fraction between 1000Da and 5000Da, adding 5%-6% of the fraction mass of trehalose, and freeze-drying to obtain the sausage pulp extract peptide.

[0016] In the above method for preparing peptides extracted from sausage pulp, the amount of deionized water used is 4 to 5 times the mass of the sausage pulp; the homogenization is performed using a homogenizer; the amount of pepsin added is 0.8% to 1.5% of the mass of the sausage pulp; the amount of trypsin added is 1.2% to 1.8% of the mass of the sausage pulp; the enzyme inactivation is performed at 85℃ to 90℃ for 10 to 20 minutes, followed by cooling to room temperature; the centrifugation is performed at 5000 r / min to 6000 r / min for 15 to 20 minutes.

[0017] The preparation method of the above-mentioned drug for preventing ischemic stroke includes the following steps:

[0018] S1 nano-coating: 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed at a mass ratio of (60-70):(30-40) to obtain a mixture; EPC and Cholesterol were mixed at a mass ratio of (58-62):(18-22) and dissolved in a chloroform-methanol mixed solvent, and then rotary evaporated to form a lipid membrane; the mixture and the lipid membrane were mixed at a mass ratio of (1.5-2):(1-1.3), hydrated with PBS, and homogenized to obtain a nano-liposome suspension with a median particle size of 100-150 nm;

[0019] S2 First-stage surface modification: Add anhydrous ethanol to DSPE-PEG2000-complex peptide to 10 mg / mL to 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution. Mix nanoliposome suspension with DSPE-PEG2000-complex peptide modification solution at a mass ratio of 10:(2~3) to obtain first-stage modification suspension.

[0020] S3 Secondary surface modification: Add anhydrous ethanol to DSPE-PEG2000-TK to 5mg / mL~8mg / mL to obtain DSPE-PEG2000-TK modification solution; Mix the primary modification suspension and DSPE-PEG2000-TK modification solution at a mass ratio of 10:(1~2) to obtain secondary modification suspension.

[0021] S4 Purification and Lyophilization: The secondary modified suspension was purified by Sepharose CL-4B agarose gel column, sterilized by a 0.22μm pore size filter membrane, and then lyophilized with trehalose after ultrafiltration to obtain the drug.

[0022] The preparation method of DSPE-PEG2000-complex peptide in the above preparation method includes: mixing CREKA peptide and sausage pulp extract peptide at a mass ratio of (50-60):(40-50), adding to PBS at pH 7.2-7.4 to a concentration of 50 mg / mL-60 mg / mL to obtain a complex peptide solution; adding DSPE-PEG2000-COOH to PBS at pH 6.0-6.2 to a concentration of 10 mg / mL-15 mg / mL, adding 10 mM-12 mM EDC and 25 mM-28 mM NHS, and activating at room temperature for 30-40 min with stirring to obtain an activation solution; adding the complex peptide solution to the activation solution at a mass ratio of DSPE-PEG2000-COOH:complex peptide = (20-25):(5-6), adjusting the pH to 7.2-7.4, and stirring at room temperature for 4-5 h; ultrafiltration through a 10 kDa ultrafiltration membrane, and taking 10 kDa... The above components were freeze-dried to obtain DSPE-PEG2000-complex peptide.

[0023] The preparation method of DSPE-PEG2000-TK in the above preparation method includes: dissolving Fmoc-TK-NH2 in a DMF solution containing 20wt% to 22wt% hexahydropyridine to a concentration of 10mg / mL to 15mg / mL, stirring at room temperature for 20min to 30min to obtain a reaction solution, adding 5 to 6 times the volume of ice-cold diethyl ether to precipitate, centrifuging at 4000r / min to 5000r / min for 10min to 15min, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 at a mass ratio of (20 to 25): (2 to 3), adding to an ethanol solution containing 40% to 50% volume of PBS to a concentration of 10mg / mL to 15mg / mL, and adding EDC. 10mM~12mM and NHS 25mM~28mM, adjust pH to 7.2~7.4, stir at room temperature for 4h~5h; ultrafiltration through a 3kDa ultrafiltration membrane, take the fraction with more than 3kDa, freeze dry to obtain DSPE-PEG2000-TK.

[0024] In the S1 nano-coating process described above, 5,7,4'-trihydroxyflavone and tetramethylpyrazine are mixed at a mass ratio of (60–70):(30–40) to obtain a mixture; EP C and Cholesterol are mixed at a mass ratio of (58–62):(18–22) and dissolved in a chloroform-methanol mixed solvent at a volume ratio of (2–2.5):1 to a concentration of 8 mg / mL–10 mg / mL. The mixture is then rotary evaporated at 38–42°C and 100–120 r / min until a uniform film is formed. After vacuum drying for 2–3 hours to remove residual solvent, a lipid membrane is obtained. The mixture and the lipid membrane are then mixed at a mass ratio of (1.5–2):(1–1.3), and 25–30 mL of the mixture is added per 1 g of lipid membrane. The ratio of PBS was adjusted, and PBS with pH 7.2 to 7.4 was added. The mixture was hydrated at 35°C to 38°C and stirred at 200 r / min to 300 r / min for 1.5 h to 2 h. The mixture was then homogenized by cycling at 1000 bar to 1200 bar for 4 to 5 times to obtain a nanoliposome suspension with a median particle size of 100 nm to 150 nm.

[0025] In the S2 primary surface modification of the above preparation method, DSPE-PEG2000-complex peptide is added to anhydrous ethanol to a concentration of 10 mg / mL to 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution; nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution are mixed at a mass ratio of 10:(2 to 3), and stirred at 80 r / min to 100 r / min for 3 to 4 hours at room temperature to complete the complex peptide modification and obtain the primary modification suspension.

[0026] In the S3 secondary surface modification of the above preparation method, DSPE-PEG2000-TK is added to anhydrous ethanol to a concentration of 5 mg / mL to 8 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension and DSPE-PEG2000-TK modification solution are mixed at a mass ratio of 10:(1 to 2), and stirred at 80 r / min to 100 r / min for 3 to 4 hours at room temperature to complete TK modification and obtain secondary modification suspension.

[0027] In the S4 purification and lyophilization process of the above preparation method, the secondary modified suspension is loaded onto a Sepharos e CL-4B agarose gel column, eluted with PBS at pH 7.2–7.4 at a flow rate of 0.3 mL / min–0.5 mL / min, and 2–3 column volumes of drug-containing eluent are collected. The eluent is then filtered through a 0.22 μm pore size filter for sterilization, and the filtrate is collected. The filtrate is then ultrafiltered through a 20 kDa ultrafiltration membrane, and the fraction with a 20 kDa or higher is taken. 5 wt%–6 wt% trehalose is added to the fraction, and the mixture is dispensed, lyophilized, and the drug is obtained.

[0028] The medication is used to prevent nerve damage after stroke.

[0029] This invention provides a drug for preventing ischemic stroke and its preparation method, with the following beneficial effects:

[0030] I. Synergistic Effects of Drug Components: 5,7,4'-Trihydroxyflavonoids (apigenin) possess antioxidant, anti-inflammatory, and anti-apoptotic properties. They can scavenge excess ROS generated during ischemia-reperfusion, reducing oxidative stress damage to nerve cells; inhibit the production and release of pro-inflammatory cytokines such as TNF-α, regulating the inflammatory response; regulate intracellular signaling pathways, inhibiting apoptosis, and promoting nerve cell survival and repair. Tetramethylpyrazine has vasodilatory, microcirculation-improving, and antiplatelet aggregation effects, increasing blood supply to ischemic areas of the brain. The two work synergistically to prevent and treat ischemic stroke.

[0031] II. Nano-encapsulation Technology: The use of EPC-Cholesterol nano-encapsulation can protect 5,7,4'-trihydroxyflavone and tetramethylpyrazine, improving drug stability and reducing decay. Simultaneously, the nanoliposomes, with a particle size of 100nm-150nm, facilitate delivery to the lesion site via blood circulation and accumulate in ischemic areas through enhanced penetration and retention effects (EPR effect).

[0032] III. Primary Modification (DSPE-PEG2000-Compound Peptide): The CREKA peptide in the compound peptide can specifically bind to fibrin at ischemic sites, giving the nanoliposomes targeting capabilities and enabling them to precisely reach the ischemic brain region; the sausage pulp-extracted peptides reduce the release of pro-inflammatory factors such as TNF-α by blocking pathways such as NF-κB, thereby alleviating the inflammatory cascade response after cerebral ischemia; and neutralize reactive oxygen species (ROS) generated during ischemia-reperfusion, thus protecting nerve cells.

[0033] CREKA peptides specifically bind to fibrin in thrombi, guiding drug accumulation in ischemic lesions. Peptides extracted from sausage pulp enhance the adhesion of nanoparticles to vascular injury sites through their cationic or amphiphilic structures; they also regulate blood-brain barrier (BBB) ​​permeability, promoting drug penetration into ischemic brain regions. A complex peptide is formed by combining these two peptides in a ratio of (50–60):(40–50), improving targeting efficiency against thrombi and inflamed vessels.

[0034] IV. Secondary Modification (DSPE-PEG2000-TK): TK represents a thioether bond, a ROS (reactive oxygen species) sensitive chemical bond. DSPE-PEG 2000 -TK modification refers to the process of adding groups containing thioether bonds through DS PE and PEG. 2000 It attaches to the surface of liposomes. In normal tissues, due to the low level of ROS, TK is shielded by PEG, reducing its toxicity to normal tissues; while in ischemic areas, the ROS level is significantly increased, which triggers PEG detachment and activates TK. The thioether bond of TK can consume local ROS and exert a synergistic antioxidant effect.

[0035] In summary, this invention achieves a synergistic effect through a scientifically designed ratio of 5,7,4'-trihydroxyflavone and tetramethylpyrazine, combined with the targeting action of CREKA peptide, the protective effect of sausage pulp-extracted peptides, and DSPE-PEG. 2000 -TK's controlled release and antioxidant effects achieve multi-level synergy of thrombus targeting, gradient controlled release, inflammation suppression, and neuroprotection, breaking through the limitations of traditional single-component drugs and improving the effectiveness of preventing neurological damage after stroke. Detailed Implementation

[0036] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0037] Definitions:

[0038] EPC: refers to egg yolk phosphatidylcholine (natural phospholipid), which, as the main component of the membrane material, can form a stable phospholipid bilayer structure.

[0039] Cholesterol: refers to cholesterol, which plays a role in regulating membrane fluidity and increasing membrane stability in liposomes.

[0040] DSPE: Distearoylphosphatidylethanolamine, a phospholipid substance used for liposome modification. It connects the modifying groups to the liposome surface by interacting with phospholipids in the liposome membrane.

[0041] PEG 2000 : indicates polyethylene glycol, with an average molecular weight of 2000 Da; PEG modification can increase the hydrophilicity of liposomes, reduce the recognition and clearance of liposomes by the mononuclear macrophage system, and prolong their time in blood circulation (i.e., play a stealth role).

[0042] CREKA peptide: This is a short peptide sequence (Cysteine-Arginine-Glutamic acid-Lysine-Alanine) that specifically binds to fibrin in the microthrombus sites of the ischemic penumbra, enabling liposomes to target and accumulate in the ischemic area. It also facilitates the crossing of the blood-brain barrier by nanoparticles, enhancing their targeting and biocompatibility. Therefore, DSPE-PEG... 2000 - Complex peptide modification involves adding DSPE and PEG. 2000 CREKA peptide and sausage pulp extract peptides are linked together through a certain chemical reaction and then modified onto the surface of liposome nanoparticles, giving liposomes the ability to target and cross the blood-brain barrier, as well as to assist in the intervention of pathological processes and maintain neurovascular function.

[0043] TK: Represents a thioether-ketal bond (TK), a type of ROS (reactive oxygen species) sensitive chemical bond. DSPE-PEG 2000 -TK modification refers to the modification of groups containing thioether bonds using DSPE and PEG. 2000 It attaches to the surface of liposomes. In normal tissues, due to the low level of ROS, TK is shielded by PEG, reducing its toxicity to normal tissues; while in ischemic areas, the ROS level is significantly increased, which triggers PEG detachment and activates TK. The thioether bond of TK can consume local ROS and exert a synergistic antioxidant effect.

[0044] Fmoc-TK-NH2: Fluorenyl methoxycarbonyl acyl-ketothiol-amino.

[0045] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0046] NHS: N-hydroxysuccinimide.

[0047] PBS: Phosphate buffer solution.

[0048] DMF: N,N-dimethylformamide, solvent.

[0049] Unit: mM: mmol / L.

[0050] Example 1

[0051] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; wherein the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are encapsulated in EPC-Cholesterol nanoparticles, followed by DSPE-PEG. 2000 - The complex peptide was modified once, followed by DSPE-PEG. 2000 -TK secondary modification yields the drug.

[0052] The preparation method of the above-mentioned drug for preventing ischemic stroke includes the following steps:

[0053] S1 nano-coating: 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed at a mass ratio of 65:35 to obtain a mixture; EPC and Cholesterol were mixed at a mass ratio of 60:20 and dissolved in a chloroform-methanol mixed solvent at a volume ratio of 2:1 to a concentration of 8 mg / mL. The mixture was then rotary evaporated at 40 °C and 100 r / min until a uniform film was formed. After vacuum drying for 2.5 h, residual solvent was removed to obtain a lipid membrane; the mixture and lipid membrane were mixed at a mass ratio of 1.5:1, and PBS at pH 7.4 was added at a ratio of 25 mL per 1 g of lipid membrane. The mixture was stirred and hydrated at 37 °C and 250 r / min for 1.5 h. After homogenization by cycling at 1000 bar for 4 times, a nanoliposome suspension with a median particle size of 122 nm was obtained.

[0054] S2 First-stage surface modification: DSPE-PEG2000-complex peptide was added to anhydrous ethanol to a concentration of 15 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution; nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution were mixed at a mass ratio of 10:2.5, and stirred at 100 r / min for 3.5 h at room temperature to complete the complex peptide modification and obtain the first-stage modified suspension;

[0055] S3 Secondary surface modification: DSPE-PEG2000-TK was added to anhydrous ethanol to a concentration of 6 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension and DSPE-PEG2000-TK modification solution were mixed at a mass ratio of 10:1.5 and stirred at 100 r / min for 3.5 h at room temperature to complete TK modification and obtain secondary modification suspension;

[0056] S4 Purification and Lyophilization: The Sepharose CL-4B agarose gel column was pre-equilibrated with pH 7.4 PBS. The secondary modification suspension was loaded onto the Sepharose CL-4B agarose gel column, with each loading volume not exceeding 1 / 10 of the column volume. Unbound modifiers and free active ingredients were adsorbed and removed. Elution was performed with pH 7.4 PBS at a flow rate of 0.4 mL / min. 2.5 column volumes of drug-containing eluent were collected, filtered through a 0.22 μm pore size filter for sterilization, and the filtrate was collected. Ultrafiltration was performed through a 20 kDa ultrafiltration membrane. The fraction with a strength of 20 kDa or higher was collected, and 5 wt% trehalose was added. The fraction was dispensed and lyophilized (lyophilization program: pre-freezing temperature -45℃, pre-freezing time 3 h, sublimation drying temperature -25℃, vacuum degree 15 Pa, sublimation drying time 15 h, desorption drying temperature 30℃, desorption drying time 8 h) to obtain the drug.

[0057] The preparation method of DSPE-PEG2000-complex peptide includes: mixing CREK A peptide and sausage pulp extract peptide at a mass ratio of 55:45, adding to pH 7.4 PBS to a concentration of 50 mg / mL to obtain a complex peptide solution; adding DSPE-PEG2000-COOH to pH 6.0 PBS to a concentration of 10 mg / mL, adding 10 mM EDC and 25 mM NHS, and activating at room temperature for 30 min to obtain an activation solution; adding the complex peptide solution to the activation solution at a mass ratio of DSPE-PEG2000-COOH:complex peptide = 20:5.5, adjusting the pH to 7.4, and stirring at room temperature for 4.5 h; ultrafiltration through a 10 kDa ultrafiltration membrane, taking the fraction with a mass greater than 10 kDa, and freeze-drying to obtain DSPE-PEG2000-complex peptide.

[0058] The preparation method of the sausage pulp extract peptides includes: adding sausage pulp to 4.5 times its weight of deionized water, homogenizing it using a homogenizer to make a pulp, adjusting the pH to 1.8, adding 1.0% of the sausage pulp weight of pepsin, and enzymatically hydrolyzing at 37℃ for 2 hours; adjusting the pH to 8.2, adding 1.5% of the sausage pulp weight of trypsin, and enzymatically hydrolyzing at 37℃ for 1 hour, inactivating the enzyme at 85℃ for 15 minutes, cooling to room temperature, centrifuging at 5000 r / min for 15 minutes, taking the supernatant, ultrafiltration using an ultrafiltration membrane, taking the fraction between 1000 Da and 5000 Da, adding 5% of the fraction weight of trehalose, and freeze-drying to obtain the sausage pulp extract peptides.

[0059] The preparation method of DSPE-PEG2000-TK includes: dissolving Fmoc-TK-NH2 in a DMF solution containing 20wt% hexahydropyridine to a concentration of 10mg / mL, stirring at room temperature for 20min to obtain a reaction solution, adding 5 times the volume of ice-cold diethyl ether (pre-cooled to 2℃) for precipitation, centrifuging at 4000r / min for 15min, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 at a mass ratio of 20:2.5, adding an ethanol solution containing 45% volume of PBS to a concentration of 10mg / mL, adding 10mM EDC and 25mM NHS, adjusting the pH to 7.4, stirring at room temperature for 4h; ultrafiltration through a 3kDa ultrafiltration membrane, taking the fraction with a concentration greater than 3kDa, and lyophilizing to obtain DSPE-PEG2000-TK.

[0060] Example 2

[0061] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; wherein the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are encapsulated in EPC-Cholesterol nanoparticles, followed by DSPE-PEG. 2000 - The complex peptide was modified once, followed by DSPE-PEG. 2000 -TK secondary modification yields the drug.

[0062] The preparation method of the above-mentioned drug for preventing ischemic stroke includes the following steps:

[0063] S1 nano-coating: 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed at a mass ratio of 60:40 to obtain a mixture; EPC and Cholesterol were mixed at a mass ratio of 58:22 and dissolved in a chloroform-methanol mixed solvent at a volume ratio of 2.2:1 to a concentration of 9 mg / mL. The mixture was rotary evaporated at 38 °C and 110 r / min until a uniform film was formed. The film was then vacuum dried for 2 h to remove residual solvent and obtain a lipid membrane. The mixture and the lipid membrane were mixed at a mass ratio of 1.7:1.2. PBS (pH 7.3) was added at a ratio of 28 mL per 1 g of lipid membrane. The mixture was stirred and hydrated at 35 °C and 200 r / min for 1.5 h. The mixture was then homogenized four times under 1100 bar pressure to obtain a nanoliposome suspension with a median particle size of 100 nm.

[0064] S2 First-stage surface modification: DSPE-PEG2000-complex peptide was added to anhydrous ethanol to a concentration of 10 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution; nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution were mixed at a mass ratio of 10:2 and stirred at 80 r / min for 3 h at room temperature to complete the complex peptide modification and obtain the first-stage modified suspension;

[0065] S3 Secondary surface modification: DSPE-PEG2000-TK was added to anhydrous ethanol to a concentration of 5 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension and DSPE-PEG2000-TK modification solution were mixed at a mass ratio of 10:1 and stirred at 80 r / min for 3 h at room temperature to complete TK modification and obtain secondary modification suspension;

[0066] S4 Purification and Lyophilization: The Sepharose CL-4B agarose gel column was pre-equilibrated with pH 7.3 PBS. The secondary modification suspension was loaded onto the Sepharose CL-4B agarose gel column, with each loading volume not exceeding 1 / 10 of the column volume. Unbound modifiers and free active ingredients were adsorbed and removed. Elution was performed with pH 7.3 PBS at a flow rate of 0.3 mL / min. Two column volumes of drug-containing eluent were collected, filtered through a 0.22 μm pore size membrane for sterilization, and the filtrate was collected. Ultrafiltration was performed through a 20 kDa ultrafiltration membrane. The fraction with a strength of 20 kDa or higher was collected, and 5.5 wt% trehalose was added. The fraction was dispensed and lyophilized (lyophilization program: pre-freezing temperature -45℃, pre-freezing time 3 h, sublimation drying temperature -25℃, vacuum degree 15 Pa, sublimation drying time 15 h, desorption drying temperature 30℃, desorption drying time 8 h) to obtain the drug.

[0067] The preparation method of DSPE-PEG2000-complex peptide includes: mixing CREK A peptide and sausage pulp extract peptide at a mass ratio of 50:50, adding to pH 7.3 PBS to a concentration of 55 mg / mL to obtain a complex peptide solution; adding DSPE-PEG2000-COOH to pH 6.1 PBS to a concentration of 12 mg / mL, adding EDC 11 mM and NHS 26 mM, stirring and activating at room temperature for 35 min to obtain an activation solution; adding the complex peptide solution to the activation solution at a mass ratio of DSPE-PEG2000-COOH:complex peptide = 22:5, adjusting the pH to 7.3, stirring and reacting at room temperature for 4 h; ultrafiltration through a 10 kDa ultrafiltration membrane, taking the fraction with a mass greater than 10 kDa, and freeze-drying to obtain DSPE-PEG2000-complex peptide.

[0068] The preparation method of the sausage pulp extract peptides includes: adding sausage pulp to 4 times its weight of deionized water, homogenizing it using a homogenizer to make a pulp, adjusting the pH to 1.5, adding 0.8% of the sausage pulp weight of pepsin, and enzymatically hydrolyzing at 36℃ for 2 hours; adjusting the pH to 8.0, adding 1.2% of the sausage pulp weight of trypsin, and enzymatically hydrolyzing at 36℃ for 1 hour, inactivating the enzyme at 88℃ for 20 minutes, cooling to room temperature, centrifuging at 5500 r / min for 20 minutes, taking the supernatant, ultrafiltration using an ultrafiltration membrane, taking the fraction between 1000 Da and 5000 Da, adding 5.5% of the fraction weight of trehalose, and freeze-drying to obtain the sausage pulp extract peptides.

[0069] The preparation method of DSPE-PEG2000-TK includes: dissolving Fmoc-TK-NH2 in a DMF solution containing 21wt% hexahydropyridine to a concentration of 12mg / mL, stirring at room temperature for 25min to obtain a reaction solution, adding 5.5 times the volume of ice-cold diethyl ether (pre-cooled to 0℃) to precipitate, centrifuging at 4500r / min for 12min, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 at a mass ratio of 23:2, adding an ethanol solution containing 40% volume of PBS to a concentration of 12mg / mL, adding 11mM EDC and 26mM NHS, adjusting the pH to 7.3, stirring at room temperature for 4.5h; ultrafiltration through a 3kDa ultrafiltration membrane, taking the fraction with a concentration greater than 3kDa, and lyophilizing to obtain DSPE-PEG2000-TK.

[0070] Example 3

[0071] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; wherein the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are encapsulated in EPC-Cholesterol nanoparticles, followed by DSPE-PEG. 2000 - The complex peptide was modified once, followed by DSPE-PEG. 2000 -TK secondary modification yields the drug.

[0072] The preparation method of the above-mentioned drug for preventing ischemic stroke includes the following steps:

[0073] S1 nano-coating: 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed at a mass ratio of 70:30 to obtain a mixture; EPC and Cholesterol were mixed at a mass ratio of 62:18 and dissolved in a chloroform-methanol mixed solvent at a volume ratio of 2.5:1 to a concentration of 10 mg / mL. The mixture was rotary evaporated at 42 °C and 120 r / min until a uniform film was formed. The film was then vacuum dried for 3 h to remove residual solvent and obtain a lipid membrane; the mixture and lipid membrane were mixed at a mass ratio of 2:1.3, and PBS at pH 7.2 was added at a ratio of 30 mL per 1 g of lipid membrane. The mixture was stirred and hydrated at 38 °C and 300 r / min for 2 h, and homogenized 5 times at 1200 bar to obtain a nanoliposome suspension with a median particle size of 150 nm.

[0074] S2 First-stage surface modification: DSPE-PEG2000-complex peptide was added to anhydrous ethanol to a concentration of 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution; nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution were mixed at a mass ratio of 10:3 and stirred at 90 r / min for 4 h at room temperature to complete the complex peptide modification and obtain the first-stage modification suspension;

[0075] S3 Secondary surface modification: DSPE-PEG2000-TK was added to anhydrous ethanol to a concentration of 8 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension and DSPE-PEG2000-TK modification solution were mixed at a mass ratio of 10:2 and stirred at 90 r / min for 4 h at room temperature to complete TK modification and obtain secondary modification suspension;

[0076] S4 Purification and Lyophilization: The Sepharose CL-4B agarose gel column was pre-equilibrated with pH 7.2 PBS. The secondary modification suspension was loaded onto the Sepharose CL-4B agarose gel column, with each loading volume not exceeding 1 / 10 of the column volume. Unbound modifiers and free active ingredients were adsorbed and removed. Elution was performed with pH 7.2 PBS at a flow rate of 0.5 mL / min. Three column volumes of drug-containing eluent were collected, filtered through a 0.22 μm pore size membrane for sterilization, and the filtrate was collected. Ultrafiltration was performed through a 20 kDa ultrafiltration membrane. The fraction with a strength of 20 kDa or higher was collected, and 6 wt% trehalose was added. The fraction was dispensed and lyophilized (lyophilization program: pre-freezing temperature -45℃, pre-freezing time 3 h, sublimation drying temperature -25℃, vacuum degree 15 Pa, sublimation drying time 15 h, desorption drying temperature 30℃, desorption drying time 8 h) to obtain the drug.

[0077] The preparation method of DSPE-PEG2000-complex peptide includes: mixing CREK A peptide and sausage pulp extract peptide at a mass ratio of 60:40, adding to pH 7.2 PBS to a concentration of 60 mg / mL to obtain a complex peptide solution; adding DSPE-PEG2000-COOH to pH 6.2 PBS to a concentration of 15 mg / mL, adding EDC 12 mM and NHS 28 mM, and activating at room temperature for 40 min to obtain an activation solution; adding the complex peptide solution to the activation solution at a mass ratio of DSPE-PEG2000-COOH:complex peptide = 25:6, adjusting the pH to 7.2, and stirring at room temperature for 5 h; ultrafiltration through a 10 kDa ultrafiltration membrane, taking the fraction with a mass greater than 10 kDa, and freeze-drying to obtain DSPE-PEG2000-complex peptide.

[0078] The preparation method of the sausage pulp extract peptides includes: adding sausage pulp to 5 times its weight of deionized water, homogenizing it using a homogenizer to make a pulp, adjusting the pH to 2.0, adding 1.5% of the sausage pulp weight of pepsin, and enzymatically hydrolyzing at 38℃ for 2.5h; adjusting the pH to 8.5, adding 1.8% of the sausage pulp weight of trypsin, and enzymatically hydrolyzing at 38℃ for 1.5h, inactivating the enzyme at 90℃ for 10min, cooling to room temperature, centrifuging at 6000r / min for 18min, taking the supernatant, ultrafiltration using an ultrafiltration membrane, taking the fraction between 1000Da and 5000Da, adding 6% of the fraction weight of trehalose, and freeze-drying to obtain the sausage pulp extract peptides.

[0079] The preparation method of DSPE-PEG2000-TK includes: dissolving Fmoc-TK-NH2 in a DMF solution containing 22wt% hexahydropyridine to a concentration of 15mg / mL, stirring at room temperature for 30min to obtain a reaction solution, adding 6 times the volume of ice-cold diethyl ether (pre-cooled to 4℃) to precipitate, centrifuging at 5000r / min for 10min, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 at a mass ratio of 25:3, adding an ethanol solution containing 50% volume of PBS to a concentration of 15mg / mL, adding 12mM EDC and 28mM NHS, adjusting the pH to 7.2, stirring at room temperature for 5h; ultrafiltration through a 3kDa ultrafiltration membrane, taking the fraction with a concentration greater than 3kDa, and lyophilizing to obtain DSPE-PEG2000-TK.

[0080] The raw materials used in the above embodiments were sourced as follows: 5,7,4'-trihydroxyflavone was from Shaanxi Yunling Biotechnology Co., Ltd., with a purity of 98%. Tetramethylpyrazine was from Shaanxi Jinkangtai Biotechnology Co., Ltd., with a purity of 98%. EPC (egg yolk phosphatidylcholine) was from Sichuan Weikeqi Biotechnology Co., Ltd. Cholesterol was from Jiangsu Yingao Biotechnology Co., Ltd., with a purity of 98%. Sepharose CL-4B agarose gel was from Fuzhou Feijing Biotechnology Co., Ltd. Trehalose was from Anhui Weimao Biotechnology Co., Ltd. CREKA peptide was from Shanghai Chutai Biotechnology Co., Ltd. DSPE-PEG2000-COOH was from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd., with a molecular weight of 2771. EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) was from Guangdong Jiudian Biotechnology Co., Ltd. NHS (N-hydroxysuccinimide) was from Shanghai Gaoming Chemical Co., Ltd., with a purity of 99%. Pepsin was from Xi'an Muguo Biotechnology Co., Ltd., with an enzyme activity of 3000 U / g. Trypsin was from [unspecified source], with an enzyme activity of 2000 U / g. Fmo c-TK-NH2 (fluorenylmethoxycarbonyl-ketothiol-amino) is sourced from Guangzhou Weihua Biotechnology Co., Ltd. Hexahydropyridine is sourced from Tianjin Jingqiang Chemical Co., Ltd. DMF (N,N-dimethylformamide) is sourced from Jinan Mingyang Chemical Co., Ltd.

[0081] Comparative Example 1

[0082] The mass ratio of 5,7,4'-trihydroxyflavone to tetramethylpyrazine was 30:70; other parameters and methods were the same as in Example 1.

[0083] Comparative Example 2

[0084] In EPC-Cholesterol, the mass ratio is EPC:Cholesterol = 70:10; other parameters and methods are the same as in Example 1.

[0085] Comparative Example 3

[0086] DSPE-PEG 2000 - In the compound peptide, the mass ratio of the compound peptide components is CREKA peptide: sausage pulp extract peptide = 30:70; other parameters and methods are the same as in Example 1.

[0087] Comparative Example 4

[0088] DSPE-PEG 2000 - In the compound peptide, all the compound peptides are CREKA peptides (without adding sausage pulp extract peptides); other parameters and methods are the same as in Example 1.

[0089] Comparative Example 5

[0090] DSPE-PEG not performed 2000 - The complex peptide was modified in one step by directly mixing the nanoliposome suspension with the DSPE-PEG2000-TK modification solution at a mass ratio of 10:1.5; other parameters and methods were the same as in Example 1.

[0091] Comparative Example 6

[0092] DSPE-PEG not performed 2000 -TK secondary modification; other parameters and methods are the same as in Example 1.

[0093] Comparative Example 7

[0094] The modification order was reversed. First, the nanoliposome suspension was mixed with DSPE-PEG2000-TK modification solution at a mass ratio of 10:1.5 to obtain a first-modified suspension. Then, the first-modified suspension was mixed with DSPE-PEG 2000-complex peptide modification solution at a mass ratio of 10:2.5 to obtain a second-modified suspension. Other parameters and methods were the same as in Example 1.

[0095] Comparative Example 8

[0096] In the preparation of peptides extracted from sausage pulp, pepsin hydrolysis was not performed; other parameters and methods were the same as in Example 1.

[0097] Comparative Example 9

[0098] In the preparation of peptides extracted from sausage pulp, trypsin hydrolysis was not performed; other parameters and methods were the same as in Example 1.

[0099] I. Acute toxicity test

[0100] Healthy ICR mice (18g-22g) were randomly assigned to 12 groups of 6 mice each, corresponding to the drugs used in each example and comparative example. The drugs were diluted with physiological saline to 2mg / mL and administered via a single tail vein injection at a dose of 40mg / kg using a 1mL sterile syringe. Mice were observed for 14 consecutive days after administration for any signs of poisoning (such as loss of appetite, lethargy, vomiting, diarrhea, convulsions, dyspnea, paralysis, etc.). On day 14, blood biochemical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr), were measured. If any abnormal blood indicators were observed, indicators of the heart, liver, spleen, lungs, and kidneys were also measured. Results: All 12 groups of mice showed no signs of poisoning, and their blood indicators were normal.

[0101] II. Animal Therapy Experiments

[0102] 1. Establishment of Experimental Animal Model: Healthy SPF-grade SD rats were randomly divided into 13 groups: 3 groups for the three examples, 9 groups for the comparative examples, and a model control group, with 6 rats in each group. An ischemic stroke model was established using the suture occlusion method: After weighing, rats were anesthetized with 3.5 mL / kg of 10% chloral hydrate via intraperitoneal injection. After successful anesthesia, the rats were fixed supine on the operating table, their necks were disinfected, and the skin was incised along the midline of the neck to separate the right common carotid artery, external carotid artery, and internal carotid artery. The distal end of the external carotid artery was ligated, and the proximal end was clamped with an arterial clamp. A small incision was made in the external carotid artery, and a suture occluded (0.22 mm diameter nylon suture with a smooth, rounded tip) was inserted into the external carotid artery. The suture occluded was then slowly advanced into the internal carotid artery to a depth of 8 mm, blocking blood flow to the middle cerebral artery and causing focal cerebral ischemia. After 120 minutes of occlusion, the suture occluded the rats to allow reperfusion. After the operation, the rats were returned to their cages and housed individually. The rats' recovery and neurological function were observed to confirm the successful establishment of the model (the rats showed signs of tilting to the opposite side).

[0103] 2. Administration method and timing: Within 1 hour of establishing the ischemic stroke model, the prepared drug in the example group and the comparative group was diluted with physiological saline to 2 mg / mL and administered via tail vein injection using a 1 mL sterile syringe at a dose of 20 mg / kg. The model control group received an equal volume of physiological saline via tail vein injection.

[0104] 3. Neurological function assessment: 48 hours after drug administration, the rats were assessed for neurological function by trained personnel who were unaware of the grouping. The scoring criteria were as follows: 0 points - no neurological deficit symptoms; 1 point - unable to fully extend the contralateral forepaw; 2 points - circling to the contralateral side; 3 points - falling to the contralateral side; 4 points - unable to walk spontaneously and loss of consciousness; the average value was taken.

[0105] 4. Measurement of Cerebral Infarction Volume: After neurological function scoring, rats were anesthetized again by intraperitoneal injection of 3.5 mL / kg of 10% chloral hydrate. Brain tissue was collected and frozen at -20℃ for 10 minutes. Using a stereotaxic instrument, the brain tissue was sliced ​​into 2 mm thick slices, a total of 5 slices. The brain slices were placed in 2% TTC solution and incubated at 37℃ in the dark for 30 minutes. Normal brain tissue was stained red, while infarcted tissue appeared white. Image-Pro Plus software was used to analyze the brain slices, calculate the area of ​​the cerebral infarction region, and multiply it by the slice thickness to obtain the cerebral infarction volume. Finally, the percentage of cerebral infarction volume to the total brain tissue volume was calculated (cerebral infarction volume percentage = cerebral infarction volume / total brain tissue volume × 100%); a range value was taken for each group.

[0106] 5. Oxidative Stress Indicators: 0.2g of brain tissue was taken and added to pre-cooled physiological saline (1:9, w / v). Homogenization was performed using a tissue homogenizer under ice bath conditions to prepare a 10% brain tissue homogenate. The homogenate was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected. Malondialdehyde (MDA) content (using the thiobarbituric acid method) and superoxide dismutase (SOD) activity (using the xanthine oxidase method) were detected using a kit. Each sample was tested in triplicate; the range values ​​were recorded for each group.

[0107] 6. Detection of inflammatory factors: Take 0.1g of brain tissue, add pre-chilled cell lysis buffer, homogenize using a tissue homogenizer under ice bath conditions, centrifuge at 12000r / min for 15 minutes at 4℃, and collect the supernatant. Detect the content of tumor necrosis factor-α (TNF-α) in brain tissue using an enzyme-linked immunosorbent assay (ELISA) kit, with 3 replicates per sample; the range value is recorded for each group.

[0108] Table 1. Results of animal treatment experiments (average values)

[0109]

[0110]

[0111] The drugs in Examples 1 to 3 all showed good therapeutic effects on indicators such as neurological function scores, percentage of cerebral infarction volume, MDA content, SOD activity, and TNF-α content. The core principle is as follows:

[0112] (1) Synergistic effect of drug components: 5,7,4'-trihydroxyflavonoids (apigenin) have antioxidant, anti-inflammatory and anti-apoptotic properties. It can clear excess ROS generated during ischemia-reperfusion, reduce the damage of oxidative stress to nerve cells; inhibit the production and release of pro-inflammatory cytokines such as TNF-α, regulate the inflammatory response; regulate intracellular signaling pathways, inhibit cell apoptosis, and promote the survival and repair of nerve cells. Tetramethylpyrazine has the effects of dilating blood vessels, improving microcirculation, and inhibiting platelet aggregation, increasing the blood supply to the ischemic area of ​​the brain. The two work synergistically to prevent and treat ischemic stroke.

[0113] (2) Nano-coating technology: EPC-Cholesterol nano-coating can protect 5,7,4'-trihydroxyflavone and tetramethylpyrazine, improve drug stability and reduce decay. At the same time, the particle size of the nanoliposomes is 100nm-150nm, which is conducive to reaching the lesion site through blood circulation and enriching in the ischemic area through enhanced penetration and retention effect (EPR effect).

[0114] (3) Primary Modification (DSPE-PEG2000-Compound Peptide): The CREKA peptide in the compound peptide can specifically bind to fibrin at ischemic sites, giving the nanoliposomes targeting capabilities and enabling them to precisely reach the ischemic brain region; the sausage pulp extract peptide reduces the release of pro-inflammatory factors such as TNF-α by blocking pathways such as NF-κB, thus alleviating the inflammatory cascade response after cerebral ischemia; it also neutralizes reactive oxygen species (ROS) generated during ischemia-reperfusion, protecting nerve cells. The sausage pulp extract peptide enhances the adhesion of nanoparticles to vascular injury sites through its cationic or amphiphilic structure, promoting drug penetration into the ischemic brain region. The combination of these two modifications to the nanoliposomes significantly improves the drug accumulation efficiency at the lesion site.

[0115] (4) Secondary modification (DSPE-PEG2000-TK): TK represents a thioether bond, a ROS (reactive oxygen species) sensitive chemical bond. DSPE-PEG 2000 -TK modification refers to the process of adding groups containing thioether bonds through DS PE and PEG. 2000 It attaches to the surface of liposomes. In normal tissues, due to the low level of ROS, TK is shielded by PEG, reducing its toxicity to normal tissues; while in ischemic areas, the ROS level is significantly increased, which triggers PEG detachment and activates TK. The thioether bond of TK can consume local ROS and exert a synergistic antioxidant effect.

[0116] In Comparative Example 1, the mass ratio of 5,7,4'-trihydroxyflavone to tetramethylpyrazine was adjusted to 30:70, resulting in a significant decrease in apigenin content. Apigenin plays a central role in the treatment of ischemic stroke; its reduction leads to: the production of large amounts of ROS and exacerbated oxidative stress after ischemic stroke. Apigenin is a strong antioxidant; insufficient levels weaken its ability to scavenge ROS, leading to increased MDA (oxidative stress products) levels, relatively decreased SOD (superoxide dismutase) activity, and aggravated oxidative damage to nerve cells. Apigenin effectively inhibits the inflammatory cascade and reduces the release of inflammatory factors such as TNF-α. With reduced levels, the inflammatory response cannot be effectively controlled, TNF-α levels rise, inflammatory damage worsens, leading to increased neurological function scores and an increased percentage of cerebral infarction volume. Because apigenin's ability to regulate intracellular signaling pathways, inhibit apoptosis, and promote nerve cell repair decreases, nerve cell survival and repair are affected, resulting in a poorer overall therapeutic effect.

[0117] In Comparative Example 2, the mass ratio of EPC to Cholesterol in the EPC-Cholesterol mixture changed to 70:10. This imbalanced ratio had multiple effects on the nanoliposomes. EPC and Cholesterol together form the lipid membrane within the nanoliposomes, and an appropriate ratio is crucial for maintaining the membrane's fluidity and stability. The altered ratio changed the structure and physical properties of the lipid membrane, making the nanoliposomes more prone to rupture in the bloodstream and leading to premature drug release. Changes in the lipid membrane affect drug loading efficiency and release rate. An inappropriate ratio reduces drug loading and makes precise control of drug release impossible, preventing effective release at ischemic sites and thus reducing the therapeutic effect, manifesting as poor neurological function recovery and large cerebral infarction areas.

[0118] In Comparative Example 3, the mass ratio of CREKA peptide to sausage pulp extract peptide in the DSPE-PEG2000-complex peptide was 30:70. Reduced targeting: The decreased proportion of CREKA peptide weakened the binding ability of nanoliposomes to fibrin in ischemic areas, making it difficult to precisely target ischemic brain regions. This reduced drug accumulation efficiency at the lesion site, hindering its therapeutic effect. While sausage pulp extract peptide and CREKA peptide exhibit a synergistic effect, this change in ratio disrupts this effect, preventing effective drug absorption or synergistic therapeutic action, thus impacting the drug's efficacy in treating ischemic stroke and leading to deterioration in various indicators.

[0119] In Comparative Example 4, the DSPE-PEG2000-complex peptide only used CREKA peptide, lacking the efficacy of sausage pulp-extracted peptides, thus failing to fully exert its protective and repairing effects on nerve cells. Relying solely on the targeting effect of CREKA peptides, without the assistance of sausage pulp-extracted peptides, the behavior of nanoliposomes in vivo is limited, making it difficult to achieve the best therapeutic effect, resulting in problems such as poor recovery of nerve function and larger cerebral infarction volume.

[0120] Comparative Example 5 skipped the initial modification with DSPE-PEG2000-complex peptide and directly underwent DSPE-PEG2000-TK modification. The initial modification with DSPE-PEG2000-complex peptide endows the nanoliposomes with targeting properties. Skipping this step prevents the nanoliposomes from specifically recognizing and binding to ischemic sites, forcing them to rely on passive diffusion to reach the lesion area. This results in a significant reduction in drug concentration at the ischemic site, greatly diminishing the therapeutic effect.

[0121] Comparative Example 6 did not undergo secondary DSPE-PEG2000-TK modification. DSPE-PEG2000-TK modification enables nanoliposomes to release drugs under the action of specific proteases at ischemic sites, achieving controlled release. Without this modification, drug release in vivo cannot be precisely controlled, resulting in premature release before reaching the lesion site or ineffective release at the lesion site, leading to reduced drug utilization and weakened therapeutic effect. DSPE-PEG 2000-TK modification also contributes to the stability of nanoliposomes in blood circulation; without this modification, the stability of nanoliposomes decreases, affecting their transport and distribution in vivo.

[0122] Comparative Example 7 reversed the modification order, performing DSPE-PEG2000-TK modification first, followed by DSPE-PEG2000-complex peptide modification. This different modification order leads to changes in the molecular arrangement and spatial structure of the nanoliposome surface. Pre-modifying with DSPE-PEG2000-TK affects the binding sites and binding efficiency of the subsequent DSPE-PEG2000-complex peptide, preventing it from effectively targeting the target and thus hindering its intended therapeutic effect in vivo.

[0123] In Comparative Example 8, the preparation of peptides extracted from sausage pulp did not involve pepsin hydrolysis. Abnormal peptide composition: Pepsin hydrolysis is a crucial step in the preparation of peptides from sausage pulp, hydrolyzing large protein molecules into peptides of specific sizes and structures. Without pepsin hydrolysis, the extracted peptide composition differs from normal, lacking key active peptides and affecting the function of the extracted peptides. The abnormal sausage pulp peptides combined with CREKA peptides form a complex peptide with reduced modification effects on nanoliposomes, leading to decreased targeting and drug absorption enhancement properties of the nanoliposomes, thus affecting the therapeutic efficacy of the drug.

[0124] In Comparative Example 9, the preparation of sausage pulp extract peptides did not involve trypsin hydrolysis. The peptide structure was incomplete: Trypsin hydrolysis further processes the peptides, giving them specific structures and activities. Without this step, the extracted sausage pulp peptides had incomplete structures and reduced activity, failing to effectively synergize with CREKA peptides. This also affected nanoliposome function: the incomplete sausage pulp extract peptides, as part of the composite peptides, could not effectively modify nanoliposomes, impacting their absorption, distribution, and targeting in vivo, ultimately reducing the therapeutic efficacy of the drug for ischemic stroke.

Claims

1. A drug for treating ischemic stroke, characterized in that, The drug comprises 5,7,4'-trihydroxyflavone and tetramethylpyrazine; the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are coated with EPC-Cholesterol nanoparticles, then modified with DSPE-PEG2000-complex peptide for one step, and then modified with DSPE-PEG2000-TK for a second step to obtain the drug. The mass ratio of the 5,7,4'-trihydroxyflavone to tetramethylpyrazine is (60-70):(30-40). In the EPC-Cholesterol mixture, the mass ratio is EPC:Cholesterol = (58-62):(18-22); In the DSPE-PEG2000-complex peptide, the mass ratio of the complex peptide components is CREKA peptide: sausage pulp extract peptide = (50-60): (40-50). The preparation method of the sausage pulp extract peptides includes: adding sausage pulp to deionized water for homogenization to make a pulp, adjusting the pH to 1.5-2.0, adding pepsin, and enzymatically hydrolyzing at 36℃-38℃ for 2-2.5 hours; adjusting the pH to 8.0-8.5, adding trypsin, and enzymatically hydrolyzing at 36℃-38℃ for 1-1.5 hours, inactivating the enzyme, centrifuging to collect the supernatant, ultrafiltration using an ultrafiltration membrane, taking the fraction between 1000 Da and 5000 Da, adding 5%-6% of the fraction mass of trehalose, and freeze-drying to obtain the sausage pulp extract peptides.

2. The drug for treating ischemic stroke according to claim 1, characterized in that, The mass ratio of the 5,7,4'-trihydroxyflavone to tetramethylpyrazine is (62-68):(32-38).

3. The drug for treating ischemic stroke according to claim 1, characterized in that, In the preparation method of peptides extracted from sausage pulp, the amount of deionized water used is 4 to 5 times the mass of sausage pulp; the homogenization is performed using a homogenizer; the amount of pepsin added is 0.8% to 1.5% of the mass of sausage pulp; the amount of trypsin added is 1.2% to 1.8% of the mass of sausage pulp; the enzyme inactivation is performed at 85℃ to 90℃ for 10 to 20 minutes, followed by cooling to room temperature; the centrifugation is performed at 5000 r / min to 6000 r / min for 15 to 20 minutes.

4. The method for preparing a drug for treating ischemic stroke according to claim 1, characterized in that, The preparation method includes the following steps: S1 nano-coating: 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed at a mass ratio of (60-70):(30-40) to obtain a mixture; EPC and Cholesterol were mixed at a mass ratio of (58-62):(18-22), dissolved in a chloroform-methanol mixed solvent, and rotary evaporated to form a lipid membrane; the mixture and the lipid membrane were mixed at a mass ratio of (1.5-2):(1-1.3), hydrated with PBS, and homogenized to obtain a nano-liposome suspension with a median particle size of 100-150 nm; S2 First-stage surface modification: Add anhydrous ethanol to DSPE-PEG2000-complex peptide to 10 mg / mL to 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution. Mix nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution at a mass ratio of 10:(2~3) to obtain a first-stage modification suspension. S3 Secondary surface modification: Add anhydrous ethanol to DSPE-PEG2000-TK to 5mg / mL~8mg / mL to obtain DSPE-PEG2000-TK modification solution; Mix the primary modification suspension and DSPE-PEG2000-TK modification solution at a mass ratio of 10:(1~2) to obtain secondary modification suspension. S4 Purification and Lyophilization: The secondary modified suspension was purified by Sepharose CL-4B agarose gel column, sterilized by a 0.22μm pore size filter membrane, and then lyophilized with trehalose after ultrafiltration to obtain the drug.

5. A method for preparing a drug for treating ischemic stroke according to claim 4, characterized in that, The preparation method of DSPE-PEG2000-complex peptide includes: mixing CREKA peptide and sausage pulp-extracted peptide at a mass ratio of (50-60):(40-50), adding to PBS at pH 7.2-7.4 to a concentration of 50 mg / mL-60 mg / mL to obtain a complex peptide solution; adding DSPE-PEG2000-COOH to PBS at pH 6.0-6.2 to a concentration of 10 mg / mL-15 mg / mL, adding 10 mM-12 mM EDC and NHS. Activate the solution by stirring at room temperature for 30-40 minutes at 25-28 mM for 30-40 minutes to obtain an activated solution; add the complex peptide solution to the activated solution at a mass ratio of DSPE-PEG2000-COOH:complex peptide = (20-25):(5-6), adjust the pH to 7.2-7.4, and stir at room temperature for 4-5 hours; ultrafilter the solution through a 10 kDa ultrafiltration membrane, take the fraction with a mass greater than 10 kDa, and freeze-dry it to obtain DSPE-PEG2000-complex peptide.

6. The method for preparing a drug for treating ischemic stroke according to claim 4, characterized in that, The preparation method of DSPE-PEG2000-TK includes: dissolving Fmoc-TK-NH2 in a DMF solution containing 20wt% to 22wt% hexahydropyridine to a concentration of 10mg / mL to 15mg / mL, stirring at room temperature for 20 to 30 minutes to obtain a reaction solution, adding 5 to 6 times the volume of ice-cold diethyl ether to precipitate, centrifuging at 4000 to 5000 r / min for 10 to 15 minutes, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 at a mass ratio of (20 to 25): (2 to 3), adding an ethanol solution containing 40% to 50% volume of PBS to a concentration of 10mg / mL to 15mg / mL, adding 10mM to 12mM EDC and NHS. 25mM~28mM, adjust pH to 7.2~7.4, stir and react at room temperature for 4h~5h; ultrafilter through a 3kDa ultrafiltration membrane, take the fraction with a value above 3kDa, and freeze dry to obtain DSPE-PEG2000-TK.

7. A method for preparing a drug for treating ischemic stroke according to claim 4, characterized in that: In S1 nano-coating, 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed at a mass ratio of (60-70):(30-40) to obtain a mixture; EPC and Cholesterol were mixed at a mass ratio of (58-62):(18-22) and dissolved in a chloroform-methanol mixed solvent at a volume ratio of (2-2.5):1 to a concentration of 8 mg / mL-10 mg / mL. The mixture was then rotary evaporated at 38-42℃ and 100-120 r / min until a uniform film was formed. After vacuum drying for 2-3 hours, residual solvent was removed to obtain a lipid membrane; the mixture and the lipid membrane were mixed at a mass ratio of (1.5-2):(1-1.3), and 25-30 mL of the mixture was added per 1 g of lipid membrane. The ratio of PBS was adjusted, and PBS with pH 7.2 to 7.4 was added. The mixture was hydrated at 35℃ to 38℃ and 200 r / min to 300 r / min for 1.5 h to 2 h. The mixture was then homogenized by cycling at 1000 bar to 1200 bar for 4 to 5 times to obtain a nanoliposome suspension with a median particle size of 100 nm to 150 nm. In the first surface modification of S2, DSPE-PEG2000-complex peptide was added to anhydrous ethanol to a concentration of 10 mg / mL to 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution; nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution were mixed at a mass ratio of 10:(2 to 3), and stirred at 80 r / min to 100 r / min for 3 to 4 hours at room temperature to complete the complex peptide modification and obtain the first modification suspension; In the secondary surface modification of S3, DSPE-PEG2000-TK was added to anhydrous ethanol to a concentration of 5 mg / mL to 8 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension and DSPE-PEG2000-TK modification solution were mixed at a mass ratio of 10:(1 to 2), and stirred at 80 r / min to 100 r / min at room temperature for 3 to 4 hours to complete TK modification and obtain secondary modification suspension; In S4 purification and lyophilization, the secondary modified suspension was loaded onto a Sepharose CL-4B agarose gel column and eluted with PBS (pH 7.2–7.4) at a flow rate of 0.3 mL / min–0.5 mL / min. Two to three column volumes of drug-containing eluent were collected, filtered through a 0.22 μm pore size membrane for sterilization, and the filtrate was collected. The filtrate was then ultrafiltered through a 20 kDa ultrafiltration membrane. Fractions with a pore size greater than 20 kDa were collected, and 5 wt%–6 wt% trehalose was added. The fractions were then aliquoted, lyophilized, and the drug was obtained.

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