Medicine for preventing cerebral arterial thrombosis and preparation method thereof

Abstract

The invention relates to a medicine for preventing cerebral arterial thrombosis and a preparation method thereof, and belongs to the technical field of biological medicine, 5, 7, 4 '-trihydroxy flavone and tetramethylpyrazine with the mass ratio of (62-68): (32-38) are sequentially subjected to EPC-Holesterone nanometer coating, primary modification of DSPE-PE G2000-composite peptide containing CREKA peptide and sausage pulp extract peptide, and secondary modification of DSPE-PEG2000-TK to obtain the medicine. According to the invention, 5, 7, 4 '-trihydroxyflavone and tetramethylpyrazine are designed to have a synergistic effect in a scientific ratio, and are matched with the targeting effect of CREKA peptide, the protective effect of sausage pulp extracted peptide and the controlled release and antioxidant effects of DSPE-PEG2000-TK, so that multi-stage synergy of thrombus targeting, gradient controlled release, inflammation inhibition and neuroprotection is realized, the limitation of a traditional single-component medicine is broken through, and the application of the medicine in the preparation of the thrombus targeted medicine is realized. The effectiveness of preventing the nerve injury after the cerebral apoplexy is improved.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomedicine technology, and specifically relates to a drug for preventing ischemic stroke and a preparation method thereof, which is a technology combining a neuroprotective drug delivery system and ischemia-reperfusion injury treatment. Background Art

[0002] Stroke, a common neurological disease that poses a serious threat to human health, is characterized by high morbidity, disability, and mortality, making it one of the leading causes of death and disability among adults worldwide. Among the many types of stroke, ischemic stroke is the most common. Its pathogenesis stems from a blood clot blocking a cerebral artery, disrupting blood supply to localized brain tissue.

[0003] During the acute phase of ischemic stroke, blood flow interruption caused by thrombosis can rapidly cause irreversible necrosis in the ischemic core, directly damaging nerve cells. During the reperfusion phase, a series of complex pathophysiological processes ensue. A massive outbreak of reactive oxygen species (ROS) leads to severe disruption of intracellular redox balance; mitochondrial dysfunction and energy metabolism disturbances occur; and the inflammatory cascade intensifies, releasing a large number of inflammatory factors, further exacerbating inflammatory damage and expanding the scope of brain injury. The interaction of these pathological processes greatly complicates the prevention and treatment of ischemic stroke.

[0004] Despite the continuous development of the medical field, the current clinical treatment effect of ischemic stroke is still unsatisfactory, and there are many pain points that need to be addressed:

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

[0006] 2. Neuroprotectants: Neuroprotectants aim to reduce brain damage and protect nerve cells through various mechanisms. However, due to the high selectivity and restrictiveness of the blood-brain barrier (BBB), most neuroprotectants have difficulty effectively penetrating the BBB and cannot achieve sufficient drug concentrations in the brain, making it difficult to exert ideal neuroprotective effects.

[0007] Given the limitations of existing treatments, finding safe and effective new approaches to prevent and treat ischemic stroke has become a top priority in medical research. In recent years, as people have become more aware of the inefficiency and severe side effects of chemical drugs, the importance of natural herbal remedies in health maintenance and disease treatment has become increasingly prominent, thanks to their diverse composition, multiple targets, and low toxicity.

[0008] Flavonoids, as important active ingredients in herbal medicines, are a well-known class of natural nutrients. They demonstrate significant potential in disease prevention and treatment due to their potent antioxidant, anti-inflammatory, and anti-apoptotic activities. Apigenin (5,7,4'-trihydroxyflavone), a typical representative of flavonoids, is widely found in a variety of herbs and plants, including celery, chamomile, and perilla. Numerous basic studies have demonstrated that apigenin possesses unique neuroprotective properties in the treatment of neurological diseases. Studies on ischemic stroke have shown that apigenin can effectively mitigate oxidative stress damage to neurons by scavenging excessive ROS. It can also inhibit the production and release of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, modulating the inflammatory response and reducing inflammatory damage to neurons. Furthermore, apigenin can modulate intracellular signaling pathways, inhibit apoptosis, and promote neuronal survival and repair.

[0009] Although apigenin has shown promising application prospects in the prevention and treatment of ischemic stroke, its clinical translation currently faces many challenges. Apigenin has poor oral availability and low bioavailability after oral absorption in the gastrointestinal tract. Its short plasma half-life and rapid metabolism and clearance in the body make it difficult to maintain effective blood concentrations. Due to its strong lipid solubility and poor water solubility, its distribution in brain tissue is extremely low, making it difficult to effectively penetrate the blood-brain barrier and reach the site of brain damage. These factors severely limit the effectiveness of apigenin in the treatment of ischemic stroke. Therefore, innovative drug delivery technologies, synergistic absorption-enhancing ingredients, and formulation optimization strategies are needed to address these issues and promote the further development of apigenin in the treatment of ischemic stroke. Summary of the Invention

[0010] Apigenin has limited utilization, low bioavailability in the gastrointestinal tract, and difficulty in effectively penetrating the blood-brain barrier, making it difficult to exert excellent preventive and therapeutic effects on ischemic stroke. The present invention provides a drug for preventing ischemic stroke and a preparation method thereof, which designs a synergistic effect of a scientific ratio of 5,7,4'-trihydroxyflavone and tetramethylpyrazine, combined with the targeting effect of CREKA peptide, the protective effect of sausage pulp extracted peptide and DSPE-PEG. 2000 -TK controlled release and antioxidant effects achieve a multi-level synergy of thrombus targeting, gradient controlled release, inflammation inhibition, and neuroprotection, breaking through the limitations of traditional single-ingredient drugs and improving the effectiveness of preventing post-stroke neurological damage. Its specific technical solutions are as follows:

[0011] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are nano-coated with EPC-Cholesterol and then nano-coated with DSPE-PEG. 2000 - One-time modification of the composite peptide followed by DSPE-PEG 2000 -TK secondary modification yields drugs.

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

[0013] In the above-mentioned 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 composite peptide, the mass ratio of the composite peptide components is CREK A peptide: sausage pulp extracted peptide = (50-60): (40-50).

[0015] In the above-mentioned composite peptide, the preparation method of the sausage pulp extracted peptide includes: adding sausage pulp to deionized water for homogenization to make pulp, adjusting the pH to 1.5-2.0, adding pepsin, and enzymatically hydrolyzing at 36°C-38°C for 2h-2.5h; adjusting the pH to 8.0-8.5, adding trypsin, and enzymatically hydrolyzing at 36°C-38°C for 1h-1.5h, inactivating the enzyme, centrifuging to obtain the supernatant, ultrafiltration using an ultrafiltration membrane, obtaining components between 1000Da and 5000Da, adding trehalose of 5%-6% by weight of the components, and freeze-drying to obtain the sausage pulp extracted peptide.

[0016] In the above-mentioned preparation method of peptide extracted from sausage pulp, the amount of deionized water used is 4 to 5 times the mass of the sausage pulp; the homogenizer is used for the homogenization; 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 is inactivated at 85°C to 90°C for 10 to 20 minutes and then cooled to room temperature; and the centrifugation is performed at 5000 r / min to 6000 r / min for 15 to 20 minutes.

[0017] The method for preparing the above-mentioned drug for preventing ischemic stroke comprises the following steps:

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

[0019] S2 primary surface modification: DSPE-PEG2000-complex peptide was added with anhydrous ethanol to 10 mg / mL to 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution. The nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution were mixed at a mass ratio of 10:(2-3) to obtain a primary modification suspension.

[0020] S3 secondary surface modification: DSPE-PEG2000-TK was added with anhydrous ethanol to 5 mg / mL to 8 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension was mixed with the DSPE-PEG2000-TK modification solution at a mass ratio of 10:(1-2) to obtain the secondary modification suspension;

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

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

[0023] In the above preparation method, the preparation method of DSPE-PEG2000-TK comprises: dissolving Fmoc-TK-NH2 in a DMF solution containing 20 wt% to 22 wt% hexahydropyridine to a concentration of 10 mg / mL to 15 mg / mL, stirring at room temperature for 20 min to 30 min to obtain a reaction solution, adding 5 to 6 times the volume of the reaction solution in glacial ether for precipitation, centrifuging at 4000 r / min to 5000 r / min for 10 min to 15 min, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 in 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 10 mg / mL to 15 mg / mL, and adding EDC. 10mM~12mM and NHS25mM~28mM, adjust the pH to 7.2~7.4, stir and react at room temperature for 4h~5h; ultrafiltration is performed through a 3kDa ultrafiltration membrane, and the fraction above 3kDa is taken and freeze-dried to obtain DSPE-PEG2000-TK.

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

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

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

[0027] In the S4 purification and lyophilization of the above preparation method, the secondary modified suspension is loaded onto a Sepharose CL-4B agarose gel column and eluted with pH 7.2-7.4 PBS at a flow rate of 0.3 mL / min to 0.5 mL / min. Two to three resin column volumes of the drug-containing eluate are collected and sterilized by passing through a 0.22 μm pore size filter membrane. The filtrate is collected and ultrafiltered through a 20 kDa ultrafiltration membrane. The fraction above 20 kDa is taken, 5 wt% to 6 wt% trehalose is added to the fraction, the fraction is aliquoted, and the fraction is lyophilized to obtain the drug.

[0028] Medications are used to prevent nerve damage after a stroke.

[0029] The present invention provides a drug for preventing ischemic stroke and a preparation method thereof, which have the following beneficial effects:

[0030] 1. Synergistic Effects of Drug Components: 5,7,4'-Trihydroxyflavone (Apigenin) has antioxidant, anti-inflammatory, and anti-apoptotic properties. It can scavenge excess ROS produced during the ischemia-reperfusion phase, alleviating oxidative stress damage to nerve cells; inhibit the production and release of proinflammatory cytokines such as TNF-α, regulating the inflammatory response; and modulate intracellular signaling pathways, inhibiting apoptosis and promoting nerve cell survival and repair. Tetramethylpyrazine has the effects of dilating blood vessels, improving microcirculation, and inhibiting platelet aggregation, increasing blood supply to ischemic areas of the brain. The two drugs work synergistically to prevent and treat ischemic stroke.

[0031] Second, nano-encapsulation technology utilizes EPC-Cholesterol nano-encapsulation to protect 5,7,4'-trihydroxyflavone and tetramethylpyrazine, improving drug stability and reducing decay. Furthermore, the nano-liposomes have a particle size of 100-150nm, facilitating their delivery to lesions through blood circulation and accumulation in ischemic areas through enhanced permeation and retention (EPR effect).

[0032] 3. Primary modification (DSPE-PEG2000-complex peptide): The CREKA peptide in the complex peptide can specifically bind to fibrin in the ischemic area, making the nanoliposome targeted and able to accurately reach the ischemic area of ​​the brain; the sausage pulp extracted peptide blocks pathways such as NF-κB, reduces the release of pro-inflammatory factors such as TNF-α, and alleviates the inflammatory cascade reaction after cerebral ischemia; neutralizes the reactive oxygen species (ROS) produced by ischemia-reperfusion, and protects nerve cells.

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

[0034] 4. Secondary modification (DSPE-PEG2000-TK): TK stands for thioether bond, which is a ROS (reactive oxygen species) sensitive chemical bond. 2000 -TK modification refers to the addition of thioether groups through DS PE and PEG 2000 Attached to the surface of liposomes. In normal tissues, due to low ROS levels, TK is shielded by PEG, reducing its toxicity to normal tissues. However, in ischemic areas, ROS levels increase significantly, triggering PEG shedding and activating TK. The thioether bond of TK can consume local ROS, exerting a synergistic antioxidant effect.

[0035] In summary, the present invention designs a synergistic effect of 5,7,4'-trihydroxyflavone and tetramethylpyrazine in a scientific ratio, cooperates with the targeting effect of CREKA peptide, the protective effect of sausage pulp extracted peptide and DSPE-PEG 2000 -TK controlled release and antioxidant effects achieve multi-level synergy of thrombus targeting-gradient controlled release-inflammation inhibition-neuroprotection, breaking through the limitations of traditional single-ingredient drugs and improving the effectiveness of preventing post-stroke nerve damage. DETAILED DESCRIPTION

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

[0037] Glossary:

[0038] EPC: refers to egg yolk phosphatidylcholine (natural phospholipid), which is the main membrane material component and 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, which connects the modification group to the liposome surface by interacting with the phospholipids in the liposome membrane.

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

[0042] CREKA peptide: It is a short peptide sequence (Cysteine-Arginine-Glutamic acid-Lysine-Alanine) that can specifically bind to fibrin in the ischemic penumbra microthrombosis, thereby enabling liposomes to target and accumulate in the ischemic area; it also helps nanoparticles pass through the blood-brain barrier, enhancing the targeting and biocompatibility of nanoparticles. 2000 -Compound peptide modification is to combine DSPE, PEG 2000 CREKA peptide and sausage pulp extracted peptide are connected through a certain chemical reaction and then modified onto the surface of liposome nanoparticles, giving the liposomes the ability to target and cross the blood-brain barrier, as well as assist in intervening in pathological processes and maintaining nerves and blood vessels.

[0043] TK: stands for Thioether-Ketal (TK), a chemical bond that is sensitive to ROS (reactive oxygen species). 2000 -TK modification refers to the addition of thioether groups via DSPE and PEG 2000 Attached to the surface of liposomes. In normal tissues, due to low ROS levels, TK is shielded by PEG, reducing its toxicity to normal tissues. However, in ischemic areas, ROS levels increase significantly, triggering PEG shedding and activating TK. The thioether bond of TK can consume local ROS, exerting a synergistic antioxidant effect.

[0044] Fmoc-TK-NH2: fluorenylmethoxycarbonyl-thioketal-amino group.

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

[0046] NHS: N-hydroxysuccinimide.

[0047] PBS: phosphate buffered saline.

[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; the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are nano-coated with EPC-Cholesterol and then nano-coated with DSPE-PEG. 2000 - One modification of the composite peptide followed by DSPE-PEG 2000 -TK secondary modification yields drugs.

[0052] The method for preparing the above-mentioned drug for preventing ischemic stroke comprises the following steps:

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

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

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

[0056] S4 purification and lyophilization: A Sepharose CL-4B agarose gel column was pre-equilibrated with pH 7.4 PBS; the secondary modified suspension was loaded onto a Sepharose CL-4B agarose gel column, with the loading volume of the secondary modified suspension not exceeding 1 / 10 of the column volume each time. Unbound modifiers and free active ingredients were removed by adsorption, and elution was performed at a flow rate of 0.4 mL / min using pH 7.4 PBS as the eluent. 2.5 resin column volumes of the drug-containing eluate were collected and sterilized by passing through a 0.22 μm pore size filter membrane. The filtrate was collected and ultrafiltered through a 20 kDa ultrafiltration membrane. Fractions above 20 kDa were taken, 5 wt% trehalose was added to the fractions, the fractions were aliquoted, and lyophilized (lyophilization procedure: pre-freezing temperature -45°C, pre-freezing time 3 h, sublimation drying temperature -25°C, vacuum degree 15 Pa, sublimation drying time 15 h, desorption drying temperature 30°C, desorption drying time 8 h) to obtain the drug.

[0057] Among them, the preparation method of DSPE-PEG2000-composite peptide includes: mixing CREK A peptide and sausage pulp extract peptide in a mass ratio of 55:45, adding to pH7.4 PBS to a concentration of 50 mg / mL to obtain a composite peptide solution; taking DSPE-PEG2000-COOH and adding it to pH6.0 PBS to a concentration of 10 mg / mL, adding EDC 10mM and NHS25mM, stirring and activating at room temperature for 30 minutes to obtain an activation solution; according to the mass ratio of DSPE-PEG2000-COOH: composite peptide = 20:5.5, adding the composite peptide solution to the activation solution, adjusting the pH to 7.4, stirring and reacting at room temperature for 4.5 hours; ultrafiltration through a 10kDa ultrafiltration membrane, taking the components above 10kDa, and freeze-drying to obtain DSPE-PEG2000-composite peptide.

[0058] The preparation method of the peptide extracted from sausage pulp includes: adding deionized water 4.5 times the mass of the sausage pulp to the sausage pulp, homogenizing with a homogenizer to make a pulp, adjusting the pH to 1.8, adding pepsin 1.0% by mass of the sausage pulp, and enzymolysis at 37°C for 2 hours; adjusting the pH to 8.2, adding trypsin 1.5% by mass of the sausage pulp, enzymolysis at 37°C for 1 hour, inactivating the enzyme at 85°C for 15 minutes, cooling to room temperature, centrifuging at 5000r / min for 15 minutes, taking the supernatant, ultrafiltration with an ultrafiltration membrane, taking the component between 1000Da and 5000Da, adding trehalose 5% by mass of the component, and freeze-drying to obtain the peptide extracted from sausage pulp.

[0059] Among them, the preparation method of DSPE-PEG2000-TK includes: taking Fmoc-TK-NH2 and dissolving it in a DMF solution containing 20wt% hexahydropyridine to a concentration of 10 mg / mL, stirring and reacting at room temperature for 20 minutes to obtain a reaction solution, adding 5 times the volume of the reaction solution of ice ether (precooled to 2°C) for precipitation, centrifuging at 4000r / min for 15 minutes, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 in a mass ratio of 20:2.5, adding to an ethanol solution containing 45% volume PBS to a concentration of 10 mg / mL, adding EDC 10mM and NHS25mM, adjusting the pH to 7.4, and stirring and reacting at room temperature for 4h; ultrafiltration through a 3kDa ultrafiltration membrane, taking the components above 3kDa, and freeze-drying to obtain DSPE-PEG2000-TK.

[0060] Example 2

[0061] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are nano-coated with EPC-Cholesterol and then nano-coated with DSPE-PEG. 2000 - One-time modification of the composite peptide followed by DSPE-PEG 2000 -TK secondary modification yields drugs.

[0062] The method for preparing the above-mentioned drug for preventing ischemic stroke comprises the following steps:

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

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

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

[0066] S4 purification and lyophilization: A Sepharose CL-4B agarose gel column was pre-equilibrated with pH 7.3 PBS; the secondary modified suspension was loaded onto a Sepharose CL-4B agarose gel column, with the loading volume of the secondary modified suspension not exceeding 1 / 10 of the column volume each time. Unbound modifiers and free active ingredients were removed by adsorption, and elution was performed at a flow rate of 0.3 mL / min using pH 7.3 PBS as the eluent. Two resin column volumes of the drug-containing eluate were collected and sterilized by passing through a 0.22 μm pore size filter membrane. The filtrate was collected and ultrafiltered through a 20 kDa ultrafiltration membrane. Fractions above 20 kDa were taken, 5.5 wt% trehalose was added to the fractions, the mixture was aliquoted, and lyophilized (lyophilization procedure: pre-freezing temperature -45°C, pre-freezing time 3 h, sublimation drying temperature -25°C, vacuum degree 15 Pa, sublimation drying time 15 h, desorption drying temperature 30°C, and desorption drying time 8 h) to obtain the drug.

[0067] Among them, the preparation method of DSPE-PEG2000-composite peptide includes: mixing CREK A peptide and sausage pulp extract peptide in a mass ratio of 50:50, adding pH7.3 PBS to a concentration of 55 mg / mL to obtain a composite peptide solution; taking DSPE-PEG2000-COOH and adding it to pH6.1 PBS to a concentration of 12 mg / mL, adding EDC 11mM and NHS26mM, stirring and activating at room temperature for 35 minutes to obtain an activation solution; according to the mass ratio of DSPE-PEG2000-COOH: composite peptide = 22:5, adding the composite peptide solution to the activation solution, adjusting the pH to 7.3, stirring and reacting at room temperature for 4 hours; ultrafiltration through a 10kDa ultrafiltration membrane, taking the components above 10kDa, and freeze-drying to obtain DSPE-PEG2000-composite peptide.

[0068] The preparation method of the peptide extracted from sausage pulp includes: adding deionized water 4 times the mass of the sausage pulp to the sausage pulp, homogenizing with a homogenizer to make a pulp, adjusting the pH to 1.5, adding pepsin in an amount of 0.8% by mass of the sausage pulp, and enzymatically hydrolyzing at 36°C for 2 hours; adjusting the pH to 8.0, adding trypsin in an amount of 1.2% by mass of the sausage pulp, and enzymatically hydrolyzing at 36°C for 1 hour, inactivating the enzyme at 88°C for 20 minutes, cooling to room temperature, centrifuging at 5500r / min for 20 minutes, taking the supernatant, ultrafiltration with an ultrafiltration membrane, taking the component between 1000Da and 5000Da, adding trehalose in an amount of 5.5% by mass of the component, and freeze-drying to obtain the peptide extracted from sausage pulp.

[0069] Among them, the preparation method of DSPE-PEG2000-TK includes: taking Fmoc-TK-NH2 and dissolving it in a DMF solution containing 21wt% hexahydropyridine to a concentration of 12 mg / mL, stirring and reacting at room temperature for 25 minutes to obtain a reaction solution, adding 5.5 times the volume of the reaction solution of ice ether (pre-cooled to 0°C) for precipitation, 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 in a mass ratio of 23:2, adding to an ethanol solution containing 40% volume PBS to a concentration of 12 mg / mL, adding EDC 11mM and NHS26mM, adjusting the pH to 7.3, and stirring and reacting at room temperature for 4.5h; ultrafiltration through a 3kDa ultrafiltration membrane, taking the components above 3kDa, and freeze-drying to obtain DSPE-PEG2000-TK.

[0070] Example 3

[0071] A drug for preventing ischemic stroke, comprising 5,7,4'-trihydroxyflavone and tetramethylpyrazine; the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are nano-coated with EPC-Cholesterol and then nano-coated with DSPE-PEG. 2000 - One modification of the composite peptide followed by DSPE-PEG 2000 -TK secondary modification yields drugs.

[0072] The method for preparing the above-mentioned drug for preventing ischemic stroke comprises the following steps:

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

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

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

[0076] S4 purification and lyophilization: A Sepharose CL-4B agarose gel column was pre-equilibrated with pH 7.2 PBS; the secondary modified suspension was loaded onto a Sepharose CL-4B agarose gel column, with the loading volume of the secondary modified suspension not exceeding 1 / 10 of the column volume each time. Unbound modifiers and free active ingredients were removed by adsorption, and elution was performed at a flow rate of 0.5 mL / min using pH 7.2 PBS as the eluent. Three resin column volumes of the drug-containing eluate were collected and sterilized by passing through a 0.22 μm pore size filter membrane. The filtrate was collected and ultrafiltered through a 20 kDa ultrafiltration membrane. Fractions above 20 kDa were taken, and 6 wt% trehalose was added to the fractions. The fractions were aliquoted and lyophilized (lyophilization procedure: pre-freezing temperature -45°C, pre-freezing time 3 h, sublimation drying temperature -25°C, vacuum degree 15 Pa, sublimation drying time 15 h, desorption drying temperature 30°C, and desorption drying time 8 h) to obtain the drug.

[0077] Among them, the preparation method of DSPE-PEG2000-composite peptide includes: mixing CREK A peptide and sausage pulp extract peptide in a mass ratio of 60:40, adding to pH7.2 PBS to a concentration of 60 mg / mL to obtain a composite peptide solution; taking DSPE-PEG2000-COOH and adding it to pH6.2 PBS to a concentration of 15 mg / mL, adding EDC 12mM and NHS28mM, stirring and activating at room temperature for 40 minutes to obtain an activation solution; according to the mass ratio of DSPE-PEG2000-COOH: composite peptide = 25:6, adding the composite peptide solution to the activation solution, adjusting the pH to 7.2, stirring and reacting at room temperature for 5 hours; ultrafiltration through a 10kDa ultrafiltration membrane, taking the components above 10kDa, and freeze-drying to obtain DSPE-PEG2000-composite peptide.

[0078] The preparation method of the peptide extracted from sausage pulp includes: adding deionized water 5 times the mass of the sausage pulp to the sausage pulp, homogenizing with a homogenizer to make a pulp, adjusting the pH to 2.0, adding pepsin 1.5% by mass of the sausage pulp, and enzymolysis at 38°C for 2.5 hours; adjusting the pH to 8.5, adding trypsin 1.8% by mass of the sausage pulp, and enzymolysis at 38°C for 1.5 hours, inactivating the enzyme at 90°C for 10 minutes, cooling to room temperature, centrifuging at 6000r / min for 18 minutes, taking the supernatant, ultrafiltration with an ultrafiltration membrane, taking the component between 1000Da and 5000Da, adding trehalose 6% by mass of the component, and freeze-drying to obtain the peptide extracted from sausage pulp.

[0079] Among them, the preparation method of DSPE-PEG2000-TK includes: taking Fmoc-TK-NH2 and dissolving it in a DMF solution containing 22wt% hexahydropyridine to a concentration of 15 mg / mL, stirring and reacting at room temperature for 30 minutes to obtain a reaction solution, adding 6 times the volume of the reaction solution of ice ether (precooled to 4°C) for precipitation, centrifuging at 5000r / min for 10 minutes, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 in a mass ratio of 25:3, adding to an ethanol solution containing 50% volume PBS to a concentration of 15 mg / mL, adding EDC 12mM and NHS28mM, adjusting the pH to 7.2, and stirring and reacting at room temperature for 5h; ultrafiltration through a 3kDa ultrafiltration membrane, taking the components above 3kDa, and freeze-drying to obtain DSPE-PEG2000-TK.

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

[0081] Comparative Example 1

[0082] The mass ratio of 5,7,4'-trihydroxyflavone to tetramethylpyrazine is 30:70; other parameters and methods are 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 composite peptide, the mass ratio of the composite peptide components is CREKA peptide: sausage pulp extracted peptide = 30:70; other parameters and methods are the same as in Example 1.

[0087] Comparative Example 4

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

[0089] Comparative Example 5

[0090] No DSPE-PEG 2000 - For single modification of the composite peptide, the nanoliposome suspension was directly mixed 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] No DSPE-PEG 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. The nanoliposome suspension was first mixed with the DSPE-PEG2000-TK modification solution at a mass ratio of 10:1.5 to obtain a primary modified suspension. The primary modified suspension was then mixed with the DSPE-PEG 2000-complex peptide modification solution at a mass ratio of 10:2.5 to obtain a secondary 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] 1. Acute toxicity test

[0100] Healthy ICR mice (18g-22g) were randomly assigned to each group of 6, for a total of 12 groups, and the drugs of each embodiment and comparative example were administered respectively. The drugs were diluted to 2 mg / mL with normal saline and administered by a single tail vein injection of 40 mg / kg using a 1mL sterile syringe; after administration, the mice were observed for 14 consecutive days for symptoms of poisoning (such as loss of appetite, slow reaction, vomiting, diarrhea, convulsions, difficulty breathing, paralysis and other abnormal symptoms). On the 14th day after administration, blood biochemical indicators, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) were tested. If there were abnormal blood indicators of poisoning, the heart, liver, spleen, lung, and kidney organ indicators were tested. Test results: All 12 groups of mice had no symptoms of poisoning, and their blood indicators were normal.

[0101] 2. Animal Therapy Experiments

[0102] 1. Establishment of the Experimental Animal Model: Healthy SPF-grade SD rats were selected and randomly divided into 13 groups, namely, 3 groups according to the 3 Examples, 9 groups according to the Comparative Examples, and a model control group, with 6 rats in each group. An ischemic stroke model was established using a suture method: the rats were weighed and anesthetized with 10% chloral hydrate (3.5 mL / kg) intraperitoneally. After successful anesthesia, the rats were fixed in a supine position on an operating table. The neck was disinfected, and the skin was incised along the midline of the neck. The right common carotid artery, external carotid artery, and internal carotid artery were isolated. The distal end of the external carotid artery was ligated, and the proximal end was clamped with an artery clamp. A small incision was made in the external carotid artery, and a suture (0.22 mm diameter nylon thread, with the tip burnt to a smooth rounded end) was inserted into the external carotid artery. The suture 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 was removed to allow reperfusion. After surgery, the rats were returned to their cages and housed individually. The rats' recovery and neurological function were observed to confirm that the model was successfully established (symptoms of falling to the contralateral side appeared).

[0103] 2. Administration Method and Schedule: Within 1 hour after establishing the ischemic stroke model, the prepared drug was diluted with normal saline to 2 mg / mL and administered via tail vein injection using a 1 mL sterile syringe at a dose of 20 mg / kg in the Example and Comparative Example groups. The Model Control group received an equal volume of normal saline injected into the tail vein.

[0104] 3. Neurological function score: 48 hours after administration, professionally trained experimenters who were unaware of the group assignments performed neurological function scores on the rats. The scoring criteria were: 0 - no neurological deficit; 1 - inability to fully extend the contralateral forepaw; 2 - turning in circles toward the contralateral side; 3 - falling toward the contralateral side; 4 - inability to walk spontaneously and loss of consciousness. The average value was used.

[0105] 4. Determination of cerebral infarction volume: After neurological function scoring, rats were anesthetized again with 10% chloral hydrate intraperitoneally at 3.5 mL / kg. The brain tissue was removed and placed in a -20°C refrigerator for 10 minutes. The brain tissue was then cut into 2 mm thick slices using a brain stereotaxic instrument, and a total of 5 slices were cut. The brain slices were placed in 2% TTC solution and incubated at 37°C in the dark for 30 minutes. Normal brain tissue was stained red, and infarcted tissue appeared white. Image-Pro Plus software was used to analyze the brain slices. The area of ​​the cerebral infarction area was calculated and then multiplied by the thickness of the brain slice 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%); the range value was used for each group.

[0106] 5. Oxidative Stress Indicator Assay: 0.2 g of brain tissue was collected and added to pre-chilled physiological saline (1:9, w / v). Homogenized in an ice bath using a tissue homogenizer 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 assayed using test kits. Three replicates were set for each sample; range values ​​were used for each group.

[0107] 6. Inflammatory Factor Detection: 0.1 g of brain tissue was collected and added to pre-chilled cell lysis buffer. The mixture was homogenized using a tissue homogenizer in an ice bath. The mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Tumor necrosis factor-α (TNF-α) levels in brain tissue were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Three replicates were performed for each sample. Range values ​​were used for each group.

[0108] Table 1 Animal treatment experimental results (average value)

[0109]

[0110]

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

[0112] (1) Synergistic effect of drug components: 5,7,4'-trihydroxyflavone (apigenin) has antioxidant, anti-inflammatory and anti-apoptotic properties. It can remove excessive ROS produced during the ischemia-reperfusion phase, 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 resisting platelet aggregation, increasing blood supply to the ischemic area of ​​the brain. The two synergistically play a role in preventing and treating ischemic stroke.

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

[0114] (3) Single modification (DSPE-PEG2000-composite peptide): The CREKA peptide in the complex peptide can specifically bind to the fibrin in the ischemic area, making the nanoliposome targeted and able to accurately reach the ischemic area of ​​the brain; the sausage pulp extracted peptide blocks the NF-κB pathway and reduces the release of pro-inflammatory factors such as TNF-α, thereby alleviating the inflammatory cascade after cerebral ischemia; it neutralizes the reactive oxygen species (ROS) generated by ischemia reperfusion and protects nerve cells. The sausage pulp extracted peptide enhances the adhesion of nanoparticles to the vascular injury site through its cationic or amphiphilic structure, promoting the penetration of drugs into the ischemic brain area. The combination of the two to modify the nanoliposome significantly improves the enrichment efficiency of the drug in the lesion site.

[0115] (4) Secondary modification (DSPE-PEG2000-TK): TK stands for thioether bond, which is a ROS (reactive oxygen species) sensitive chemical bond. 2000 -TK modification refers to the addition of thioether groups through DS PE and PEG 2000 Attached to the surface of liposomes. In normal tissues, due to low ROS levels, TK is shielded by PEG, reducing its toxicity to normal tissues. However, in ischemic areas, ROS levels increase significantly, triggering PEG shedding and activating TK. The thioether bond of TK can consume local ROS, exerting a synergistic antioxidant effect.

[0116] In Comparative Example 1, the mass ratio of 5,7,4'-trihydroxyflavone to tetramethylpyrazine was adjusted to 30:70, and the apigenin content was significantly reduced. Apigenin plays a core role in the treatment of ischemic stroke. A reduction in its content will result in: after ischemic stroke occurs, a large amount of ROS is produced, and oxidative stress is aggravated. Apigenin is a strong antioxidant. Its insufficient content weakens the ability to clear ROS, resulting in an increase in MDA (oxidative stress product) content, a relative decrease in SOD (antioxidant enzyme) activity, and increased oxidative damage to nerve cells. Apigenin can effectively inhibit the inflammatory cascade and reduce the release of inflammatory factors such as TNF-α. After its content is reduced, the inflammatory response cannot be effectively controlled, the TNF-α content increases, and the inflammatory damage is aggravated, which leads to an increase in neurological function score and an increase in the percentage of cerebral infarction volume. Due to the decreased ability of apigenin to regulate intracellular signaling pathways, inhibit cell apoptosis and promote nerve cell repair, the survival and repair of nerve cells are affected, making the overall therapeutic effect worse.

[0117] In comparative example 2, EPC and Cholesterol mass ratio become 70:10 in EPC-Cholesterol, and this imbalance of proportions produces many-sided influence on nano liposome.EPC and Cholesterol constitute lipid membrane together in nano liposome, and suitable ratio is most important for maintaining the fluidity, stability of lipid membrane.After ratio changes, the structure and physical property of lipid membrane change, cause nano liposome to break easily in blood circulation, and the drug carried is released in advance.The variation of lipid membrane affects the carrying efficiency and the release rate of drug.Unsuitable ratio reduces the drug carrying capacity, and drug release cannot be accurately controlled, can not effectively release at ischemic site, thereby reduces the therapeutic effect of drug, shows as poor recovery of neurological function, large area of ​​cerebral infarction etc.

[0118] In Comparative Example 3, the mass ratio of CREKA peptide to salami pulp peptide in the DSPE-PEG2000 composite peptide was 30:70. Reduced targeting: The reduced proportion of CREKA peptide weakened the nanoliposome's ability to bind to fibrin in the ischemic area, making it difficult to accurately target the ischemic area of ​​the brain. This reduced drug accumulation efficiency at the lesion site and inability to fully exert its therapeutic effect. The salami pulp peptide and CREKA peptide exhibit a synergistic effect. Changing the ratio disrupted this synergistic effect, making it impossible to effectively promote drug absorption or exert a synergistic therapeutic effect, thereby affecting the drug's therapeutic effect on ischemic stroke and causing various indicators to deteriorate.

[0119] In Comparative Example 4, the DSPE-PEG2000-composite peptide, which only uses CREKA peptide and lacks the efficacy of the chorizo ​​pulp peptide, cannot fully exert its protective and repair effects on nerve cells. Relying solely on the targeting effect of CREKA peptide, without the support of the chorizo ​​pulp peptide, the nanoliposomes' behavior in the body is limited, making it difficult to achieve optimal therapeutic effects. This leads to problems such as poor neurological recovery and larger cerebral infarction volume.

[0120] Comparative Example 5 did not undergo the primary modification with the DSPE-PEG2000-complex peptide, but directly performed the DSPE-PEG2000-TK modification. The primary modification with the DSPE-PEG2000-complex peptide imparts targeting to the nanoliposomes. However, after skipping this step, the nanoliposomes cannot specifically recognize and bind to the ischemic site and can only rely on passive diffusion to reach the lesion area, resulting in a significant decrease in the concentration of the drug in the ischemic site and a significant reduction in the therapeutic effect.

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

[0122] Comparative Example 7 reverses the modification order, first performing DSPE-PEG2000-TK modification and then DSPE-PEG2000-composite peptide modification. Different modification orders will lead to changes in the molecular arrangement and spatial structure on the surface of the nanoliposomes. Performing DSPE-PEG2000-TK modification first will affect the binding site and binding efficiency of the subsequent DSPE-PEG2000-composite peptide, making it impossible for the complex peptide to effectively exert its targeting effect, resulting in it not being able to function as expected in the body, thereby reducing the therapeutic effect.

[0123] In Comparative Example 8, the preparation of the sausage pulp-extracted peptides was performed without pepsin hydrolysis. The peptide composition was abnormal: Pepsin hydrolysis is a key step in the preparation of the sausage pulp-extracted peptides, which can hydrolyze macromolecular proteins into peptides of specific size and structure. Without pepsin hydrolysis, the composition of the extracted peptides was different from that of the normal ones, lacking key active peptides, which affected the function of the sausage pulp-extracted peptides. The composite peptide composed of the abnormal sausage pulp-extracted peptide and the CREKA peptide had a poor modification effect on the nanoliposomes, resulting in reduced performance of the nanoliposomes, such as targeting and drug absorption promotion, thereby affecting the therapeutic effect of the drug.

[0124] In Comparative Example 9, the preparation of the sausage pulp extracted peptide was not subjected to trypsin hydrolysis. The peptide structure is incomplete: trypsin hydrolysis further processes the peptide to give it a specific structure and activity. Without this step, the extracted sausage pulp extracted peptide structure is incomplete, the activity is reduced, and it cannot effectively synergize with the CREKA peptide. Affecting the function of nanoliposomes: The composite peptide composed of incomplete sausage pulp extracted peptides cannot effectively modify the nanoliposomes, resulting in the absorption, distribution and targeting of the nanoliposomes in the body being affected, and ultimately reducing the therapeutic effect of the drug on ischemic stroke.

Claims

1. A drug for preventing ischemic stroke, characterized in that: The drugs include 5,7,4'-trihydroxyflavone and tetramethylpyrazine; the 5,7,4'-trihydroxyflavone and tetramethylpyrazine are nano-coated with EPC-Cholesterol and then nano-coated with DSPE-PEG. 2000 - One-time modification of the composite peptide followed by DSPE-PEG 2000 -TK secondary modification yields drugs.

2. The drug for preventing 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 preventing ischemic stroke according to claim 1, characterized in that: In the EPC-Cholesterol, the mass ratio is EPC:Cholesterol=(58-62):(18-22).

4. The drug for preventing ischemic stroke according to claim 1, wherein The DSPE-PEG 2000 - In the composite peptide, the mass ratio of the composite peptide components is CREKA peptide: sausage pulp extracted peptide = (50-60): (40-50).

5. The drug for preventing ischemic stroke according to claim 4, characterized in that: The preparation method of the sausage pulp extracted peptide comprises the following steps: adding the sausage pulp to deionized water for homogenization to obtain pulp, adjusting the pH value to 1.5-2.0, adding pepsin, and performing enzymatic hydrolysis at 36-38°C for 2-2.5 hours; adjusting the pH value to 8.0-8.5, adding trypsin, and performing enzymatic hydrolysis at 36-38°C for 1-1.5 hours, inactivating the enzyme, centrifuging to obtain a supernatant, performing ultrafiltration using an ultrafiltration membrane, obtaining components between 1000Da and 5000Da, adding trehalose in an amount of 5%-6% by weight of the components, and freeze-drying to obtain the sausage pulp extracted peptide.

6. The drug for preventing ischemic stroke according to claim 5, characterized in that: The amount of deionized water used is 4 to 5 times the mass of the sausage pulp; the homogenizer is used for the homogenization; 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 is inactivated at 85°C to 90°C for 10 to 20 minutes and then cooled to room temperature; the centrifugation is performed at 5000 r / min to 6000 r / min for 15 to 20 minutes.

7. A method for preparing a drug for preventing ischemic stroke, for preparing the drug for preventing ischemic stroke according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: S1 nanocoating: 5,7,4'-trihydroxyflavone and tetramethylpyrazine were mixed in a mass ratio of (60-70):(30-40) to obtain a mixture; EPC and Cholesterol were mixed in a mass ratio of (58-62):(18-22), dissolved in a chloroform-methanol mixed solvent, and rotary evaporated to form a lipid film; the mixture and the lipid film were mixed in a mass ratio of (1.5-2):(1-1.3), hydrated with PBS, and homogenized to obtain a nanoliposome suspension with a median particle size of 100-150 nm; S2 primary surface modification: DSPE-PEG2000-complex peptide was added with anhydrous ethanol to 10 mg / mL to 20 mg / mL to obtain DSPE-PEG2000-complex peptide modification solution. The nanoliposome suspension and DSPE-PEG2000-complex peptide modification solution were mixed at a mass ratio of 10:(2-3) to obtain a primary modification suspension. S3 secondary surface modification: DSPE-PEG2000-TK was added with anhydrous ethanol to 5 mg / mL to 8 mg / mL to obtain DSPE-PEG2000-TK modification solution; the primary modification suspension was mixed with the DSPE-PEG2000-TK modification solution at a mass ratio of 10:(1-2) to obtain the secondary modification suspension; S4 purification and lyophilization: The secondary modified suspension was purified by Sepharose CL-4B agarose gel column, sterilized by 0.22 μm pore size filter membrane, ultrafiltered, and lyophilized with trehalose to obtain the drug.

8. The method for preparing a drug for preventing ischemic stroke according to claim 7, characterized in that: The preparation method of DSPE-PEG2000-composite peptide comprises the following steps: mixing CREKA peptide and sausage pulp extract peptide in a mass ratio of (50-60): (40-50), adding the mixture to pH 7.2-7.4 PBS to a concentration of 50 mg / mL-60 mg / mL to obtain a complex peptide solution; adding DSPE-PEG2000-COOH to pH 6.0-6.2 PBS to a concentration of 10 mg / mL-15 mg / mL, and adding EDC. 10mM~12mM and NHS25mM~28mM, stir and activate at room temperature for 30min~40min to obtain an activation solution; according to the mass ratio of DSPE-PEG2000-COOH: composite peptide = (20~25):(5~6), add the composite peptide solution to the activation solution, adjust the pH to 7.2~7.4, stir and react at room temperature for 4h~5h; ultrafiltration is performed through a 10kDa ultrafiltration membrane, and the components above 10kDa are taken and freeze-dried to obtain DSPE-PEG2000-complex peptide.

9. The method for preparing a drug for preventing ischemic stroke according to claim 7, wherein: The preparation method of DSPE-PEG2000-TK includes: dissolving Fmoc-TK-NH2 in a DMF solution containing 20 wt% to 22 wt% hexahydropyridine to a concentration of 10 mg / mL to 15 mg / mL, stirring at room temperature for 20 min to 30 min to obtain a reaction solution, adding 5 to 6 times the volume of the reaction solution in glacial ether for precipitation, centrifuging at 4000 r / min to 5000 r / min for 10 min to 15 min, taking the precipitate, and vacuum drying to constant weight to obtain TK-NH2; mixing DSPE-PEG2000-COOH and TK-NH2 in a mass ratio of (20 to 25): (2 to 3), adding the mixture to an ethanol solution containing 40% to 50% by volume of PBS to a concentration of 10 mg / mL to 15 mg / mL, and adding EDC. 10mM~12mM and NHS25mM~28mM, adjust the pH to 7.2~7.4, stir and react at room temperature for 4h~5h; ultrafiltration is performed through a 3kDa ultrafiltration membrane, and the fraction above 3kDa is taken and freeze-dried to obtain DSPE-PEG2000-TK.

10. The method for preparing a drug for preventing ischemic stroke according to claim 7, characterized in that: In the S1 nanocoating, 5,7,4'-trihydroxyflavone and tetramethylpyrazine are mixed in a mass ratio of (60-70):(30-40) to obtain a mixture; EPC and Cholesterol are mixed in a mass ratio of (58-62):(18-22), dissolved in a chloroform-methanol mixed solvent with a volume ratio of (2-2.5):1 to a concentration of 8 mg / mL-10 mg / mL, and rotary evaporated at 38°C-42°C and 100 r / min-120 r / min to form a uniform film, vacuum dried for 2h-3h, and the residual solvent is removed to obtain a lipid film; the mixture and the lipid film are mixed in a mass ratio of (1.5-2):(1-1.3), and 25mL-30mL is added per 1g of lipid film. The mixture was stirred at 35°C to 38°C and 200-300 r / min for 1.5-2 hours, and the mixture was homogenized 4-5 times under a pressure of 1000-1200 bar to obtain a nanoliposome suspension with a median particle size of 100-150 nm. In the primary surface modification of S2, DSPE-PEG2000-composite peptide was added to anhydrous ethanol to a concentration of 10 mg / mL to 20 mg / mL to obtain a DSPE-PEG2000-composite peptide modification solution; the nanoliposome suspension and the DSPE-PEG2000-composite peptide modification solution were mixed in a mass ratio of 10:(2-3), and the mixture was stirred at 80 rpm to 100 rpm for 3 h to 4 h at room temperature to complete the complex peptide modification and obtain a primary 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 a DSPE-PEG2000-TK modification solution; the primary modification suspension was mixed with the DSPE-PEG2000-TK modification solution in a mass ratio of 10:(1-2), and the mixture was stirred at 80 rpm to 100 rpm at room temperature for 3 h to 4 h to complete the TK modification and obtain a secondary modification suspension; In the S4 purification and lyophilization, the secondary modified suspension is loaded onto a Sepharose CL-4B agarose gel column and eluted with pH 7.2-7.4 PBS at a flow rate of 0.3 mL / min-0.5 mL / min. Two to three resin column volumes of the drug-containing eluate are collected and sterilized by passing through a 0.22 μm pore size filter membrane. The filtrate is collected and ultrafiltered through a 20 kDa ultrafiltration membrane. The fraction above 20 kDa is taken, 5 wt%-6 wt% trehalose is added to the fraction, the fraction is aliquoted, and the fraction is lyophilized to obtain the drug.

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