Use of a polypeptide combined with Panax notoginseng for treating cardiovascular and cerebrovascular embolic diseases

Abstract

The present invention provides a use of a polypeptide and Panax notoginseng in combination for treating cardiovascular and cerebrovascular embolic diseases, belonging to the field of biomedicine. Using Mytilus fusca as a raw material, the present invention uses mixed protease enzymatic hydrolysis, separation, and purification to obtain a polypeptide with therapeutic effects on cardiovascular and cerebrovascular embolism. The polypeptide, when used in combination with Panax notoginseng, significantly enhances the therapeutic effect on embolism while being non-toxic to normal cells. The polypeptide can be used to prevent and treat thrombosis and embolic diseases, providing a reference for the research and development of a novel therapeutic drug for cardiovascular and cerebrovascular diseases and possessing broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a use of a polypeptide and Panax notoginseng in combination for treating cardiovascular and cerebrovascular embolic diseases. Background Art

[0002] Cardiovascular disease (CVD) is a general term for cardiovascular and cerebrovascular diseases, encompassing ischemic or hemorrhagic disorders of the heart, brain, and systemic tissues caused by conditions such as hyperlipidemia, blood viscosity, atherosclerosis, and hypertension. The "China Cardiovascular Disease Report 2016" indicates that CVD is the leading cause of death in China, posing a serious threat to human health and quality of life. Research into therapeutics for CVD is of immense social need and significance. When blood flow is unhealthy, small deposits, or thrombi, form on the surface of debonded or repaired vessels within the cardiovascular system. In variable flow-dependent patterns, thrombi are composed of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells, which can compromise overall health.

[0003] In general, anticoagulant, antithrombotic and anti-inflammatory treatments play an important role in the management of cardiovascular diseases. However, the safety and efficacy of these drugs limit their clinical application and are often accompanied by complications such as hemorrhagic bullous dermatitis, intramedullary hemorrhage, thrombocytopenia, etc. Therefore, it is of great significance to find and research and develop new active ingredients with high efficiency and low toxicity for the treatment of cardiovascular diseases.

[0004] Modern pharmacological studies have shown that the main active ingredients of animal-based Chinese medicines are small peptides, which have multiple biological and pharmacological activities, such as antibacterial, anti-inflammatory, antioxidant, and immunomodulatory. They can exert their effects by binding to cell surface receptors, regulating signal transduction pathways, and inhibiting enzyme activity. Studies have shown that small peptides in animal-based medicinal materials have good bioavailability and efficacy, and can be better absorbed and utilized by the human body. Therefore, these small peptides are widely used in the field of traditional Chinese medicine and have become one of the hot spots in modern drug research.

[0005] Chinese invention patent CN118791569A discloses the use of two deep-sea coagulation factor XIa inhibitory peptides, PRNIF and GNDRCL, and their compositions in the preparation of anticoagulants, antithrombotic drugs or compositions. The polypeptide is derived from the deep-sea flat-ended clam, and is obtained by virtual enzymatic hydrolysis of the deep-sea flat-ended clam protein sequence, and then obtained after virtual screening with coagulation factor XIa as the target. Studies have shown that both polypeptides, PRNIF and GNDRCL, have good coagulation factor XIa (FXIa) inhibitory activity, with IC50 values ​​of 0.67mM and 1.52mM, respectively. Double reciprocal plotting shows that PRNIF is a non-competitive inhibitor. In in vitro anticoagulation experiments, PRNIF can significantly prolong the thrombin time (TT) and activated partial thromboplastin time (APTT), and has a significant anticoagulant effect. The two anticoagulant peptides provided by the present invention are derived from deep-sea shellfish, have novel sequences and low molecular weight, can be used as template molecules for modification and transformation for the research and development of peptide anticoagulant drugs or antithrombotic drugs, and have good development and utilization prospects.

[0006] Chinese invention patent CN116082440A discloses a tuna bone antioxidant oligopeptide and its preparation method. The tuna bone antioxidant oligopeptide has good scavenging activity, thermal stability, and digestibility. Its mechanism of action is to promote the release of nitric oxide (NO), increase the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), and total antioxidant capacity (T-AOC), and reduce the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH). JQYG-1 can protect HUVEC cells from hydrogen peroxide-induced oxidative damage. By exerting its antioxidant effects and protecting vascular endothelial cells, JQYG-1 may provide a candidate drug for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, and heart failure.

[0007] The present invention uses thick-shelled mussels, which are widely sourced from the ocean, as raw materials to extract small molecule active peptides from the mussel meat, and combines them with Panax notoginseng to treat cardiovascular and cerebrovascular embolic diseases, providing new ideas for the utilization of marine resources and the treatment of embolic diseases. SUMMARY OF THE INVENTION

[0008] In order to solve the problems existing in the prior art, the present invention provides a use of a polypeptide in combination with Panax notoginseng for treating cardiovascular and cerebrovascular embolic diseases.

[0009] To this end, the present invention adopts the following technical solutions:

[0010] The present invention provides a composition for treating cardiovascular and cerebrovascular embolic diseases. The composition consists of a polypeptide and Panax notoginseng.

[0011] Furthermore, the polypeptide is selected from:

[0012] (1) a polypeptide having an amino acid sequence as shown in any one of SEQ ID No. 1-2; and / or

[0013] (2) A polypeptide having an amino acid sequence homology of at least 70% with any one of SEQ ID No. 1-2 and having the same or similar function.

[0014] Furthermore, the polypeptide is a polypeptide having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% homology to the amino acid sequence shown in any one of SEQ ID No. 1-2, and having the same or similar function.

[0015] Furthermore, the amino acid sequences of the polypeptides are shown in SEQ ID NO. 1 (NGSGKYLTNF) and SEQ ID NO. 2 (KMWTAIKKGEL).

[0016] Furthermore, the polypeptide is derived from Mytilus edulis.

[0017] The present invention also provides a pharmaceutical composition comprising the composition or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0018] The pharmaceutical excipients described herein can be those widely used in the pharmaceutical production field. Excipients are primarily used to provide a safe, stable, and functional pharmaceutical composition. They can also provide methods to dissolve the active ingredient at a desired rate after administration to a subject, or to promote effective absorption of the active ingredient after administration of the composition to a subject. The pharmaceutical excipients described herein can be inert fillers, or can provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. The pharmaceutical excipients described herein can include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0019] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0020] Furthermore, the dosage forms of the pharmaceutical composition include tablets, capsules, granules, pills, powders, suspensions, emulsions, drops, and mixtures.

[0021] The pharmaceutical compositions of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ophthalmic, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0022] The present invention also provides the use of the polypeptide and Panax notoginseng in preparing a medicine for treating cardiovascular and cerebrovascular embolic diseases.

[0023] The present invention also provides a composition and an application of the pharmaceutical composition in the preparation of antithrombotic drugs.

[0024] Furthermore, the drug is used to treat deep vein thrombosis, thromboembolic disease, thrombotic cerebral infarction, pulmonary embolism, or acute coronary syndrome.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] The present invention extracts polypeptides with amino acid sequences such as SEQ ID NO.1 (NGSGKYLTNF) and SEQID NO.2 (KMWTAIKKGEL) from thick-shelled mussels, which can prolong the activated partial thromboplastin time of rabbits in a concentration-dependent manner. In addition, in the process of combined use with Panax notoginseng powder, it can significantly improve brain edema, reduce cerebral vascular permeability, and reduce the amount of Evans blue exudation in brain tissue, and has a significant blood-brain barrier protection effect; it can reduce the NOS and MDA levels in the brain of ischemic rats, and has a protective effect on brain damage caused by ischemia. On the basis that the mussel polypeptide and Panax notoginseng powder of the present invention have synergistic effects, the present invention can be used to prevent and treat thrombosis and embolism diseases, provides a reference for the research and development of a new therapeutic drug for cardiovascular and cerebrovascular diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. Graph showing the cytotoxic effects of the polypeptides of the present invention.

[0028] Figure 2 . A graph showing the effect of the polypeptide of the present invention on the activated partial thromboplastin time (APPT) of rabbits. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific examples. Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0030] It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0031] Example 1 Preparation of polypeptide

[0032] (1) Clean fresh thick-shelled mussels, remove the shells and internal organs, take the mussel meat, wash and drain it, and grind it into minced meat in a meat grinder;

[0033] (2) adding 6 times the amount of water to the minced meat according to the weight ratio, and then homogenizing in a dispersing homogenizer for 5 minutes to obtain a mussel meat homogenate;

[0034] (3) adjusting the pH of the mussel meat homogenate obtained in step (2) to 7.0, boiling for 20 minutes, cooling to room temperature, adjusting the pH to 2.0, adding 1% pepsin, and enzymolyzing at 40°C for 2 hours; adjusting the pH to 7.0, adding 2% trypsin, and enzymolyzing at 40°C for 2 hours; and simultaneously performing ultrasonic treatment during the enzymolysis process;

[0035] (4) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis reaction solution is kept at 95°C for 100 minutes to inactivate the enzyme and then cooled to room temperature;

[0036] (5) taking the room temperature enzymatic hydrolysis reaction solution of step (4) and centrifuging it at 6000 rpm for 30 min, collecting the supernatant to obtain the mussel enzymatic hydrolysate;

[0037] (6) the supernatant mussel hydrolysate is ultrafiltered through a 3KD ultrafiltration membrane, and desalted by electrodialysis or dialysis bag to obtain a polypeptide solution;

[0038] (7) The polypeptide solution is separated by SP-650M ion exchange column and then separated by Sephadex G-50 gel chromatography; further separation is performed by RP-HPLC reverse phase high performance liquid chromatography, and the components are collected and freeze-dried for later use to obtain a high-purity polypeptide;

[0039] (8) The peptide was subjected to alternating primary and secondary mass spectrometry using a mass spectrometer, and analyzed with the aid of software to obtain two peptide amino acid sequences, as shown in SEQ ID NO. 1 (NGSGKYLTNF) and SEQ ID NO. 2 (KMWTAIKKGEL), respectively. The peptide was commissioned to be synthesized by Shanghai Bioengineering for future use.

[0040] Example 2: Evaluation of Peptide Toxicity

[0041] Test samples: polypeptides represented by SEQ ID NO. 1 (NGSGKYLTNF) and SEQ ID NO. 2 (KMWTAIKKGEL) prepared in Example 1.

[0042] Experimental method: HeLa cells grown to 80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin and cultured with complete culture medium to a cell density of 1×10 5 / mL of cell suspension. 100 μL of cell suspension was inoculated into a 96-well culture plate and placed in an incubator at 37°C and 5% CO2 saturated humidity. After 24 hours of cell culture, the complete culture medium was aspirated. Mussel polypeptides diluted with high-glucose DMEM medium at concentrations of 10 μg / ml and 100 μg / ml were added to the experimental group (3 replicates were set for each concentration), the control group was cells cultured in DMEM medium, and the blank group was DMEM medium without cells, and continued to be cultured in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours. 10 μL of CCK-8 reagent was added to each group, incubated in a cell culture incubator for 2 hours, and the absorbance value (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay. The cell survival rate was calculated according to the following formula based on the mean absorbance of each group:

[0043]

[0044] Test results: The test results are as follows: Figure 1 As shown, the cell viability of each experimental group was above 100%, indicating that the mussel polypeptides NGSGKYLTNF and KMWTAIKKGEL prepared by the present invention had no cytotoxicity and good safety.

[0045] Example 3 Effect of mussel polypeptide on activated partial thromboplastin time (APPT)

[0046] The activated partial thromboplastin time (APPT) test is mainly used to evaluate the effect of drugs on the intrinsic coagulation pathway. 2+ Fibrinogen is converted into insoluble fibrin under the participation of the plasma thromboplastin, and the time required for coagulation is measured, which is the activated partial thromboplastin time of the plasma to be tested.

[0047] The specific test procedure was performed according to the kit instructions (01020138, Sun Biotechnology), and clotting time was measured using a semi-automated coagulation analyzer (HF6000-4, Jinan Hanfang). The procedure was as follows: magnetic beads were added to each coagulation cup. 30 μL of APTT reagent, 30 μL of citrated rabbit plasma, and 10 μL of peptides SEQ ID NO. 1 (NGSGKYLTNF) and SEQ ID NO. 2 (KMWTAIKKGEL) were mixed at 37°C and added to the cup. After incubation at 37°C for 1 minute, 30 μL of preheated 25 mM CaCl2 solution was added, and the coagulation time was measured.

[0048] The experimental results are as follows Figure 2 As shown, mussel polypeptides SEQ ID NO. 1 (NGSGKYLTNF) and SEQ ID NO. 2 (KMWTAIKKGEL) can prolong rabbit APPT in a concentration-dependent manner.

[0049] Example 4 Effect of combined use of mussel polypeptide and Panax notoginseng

[0050] (1) Experimental animals: 96 SPF-grade SD rats, male, 6-8 weeks old, weighing 200-150 g, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd., with the experimental animal production license number: SCXK (Yue) 2022-0063. They were quarantined for 3 days and used after no abnormalities were found.

[0051] (2) Test samples: mussel polypeptide prepared in Example 1, Panax notoginseng powder (manufacturer: Beijing Tongrentang (Bozhou) Medicinal Pieces Co., Ltd.), positive control: Naoxintong capsule (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number 171285), all of the above drugs were prepared into solutions of the required concentration with distilled water and set aside.

[0052] (3) Establishment of an acute incomplete cerebral ischemia model: SD rats were anesthetized with 20% urethane intraperitoneally and fixed in a supine position on a surgical board. The neck was disinfected with iodine, and the common carotid arteries were surgically isolated. Except for the normal control group, which underwent sham surgery, the common carotid arteries of all other groups were ligated bilaterally and covered with sterile gauze.

[0053] (4) Grouping and administration: Male SD rats that passed the quarantine were randomly divided into groups according to weight, with 8 rats in each group. The test drugs were administered by gavage for 10 consecutive days.

[0054] Table 1 Grouping and Dosage Table

[0055]

[0056] (5) Detection method

[0057] 1. Determination of Cerebral Vascular Permeability

[0058] One hour after the last administration, the model was established. Evans blue was injected into the femoral vein of rats in the normal control group without ligating the common carotid arteries. Rats in the remaining groups were injected with Evans blue via the femoral vein 5 minutes before ligating the common carotid arteries. After ligation, rats in each group were sacrificed by cervical dislocation. The brain was removed through craniotomy, rinsed with saline, blotted dry with filter paper, and weighed. The brain tissue was immersed in 5 ml of formamide and incubated in a 45°C incubator for 72 hours. The supernatant was then measured with a spectrophotometer at a wavelength of 620 nm.

[0059] 2. Brain Index and Brain Water Content Determination

[0060] One hour after the last administration, the model was established and rats in each group were killed by cervical dislocation. The brain was removed by craniotomy and rinsed with saline, blotted dry with filter paper, and the left and right brain tissues were cut and weighed separately. The left brain tissue was then placed in an oven and dried at 180°C to constant weight, and then weighed. The brain index and brain water content were calculated according to the following formula:

[0061] Brain index = brain mass / body mass x 100%; brain water content = (wet mass - dry mass) / wet mass x 100%.

[0062] 3. Determination of nitric oxide synthase (NOS) and malondialdehyde (MDA) in brain tissue

[0063] The right brain was weighed and placed in a glass homogenizer. 5 ml of normal saline was added to prepare a brain homogenate. The homogenate was centrifuged at 3000 rpm for 10 minutes. The supernatant was placed in an ice bath. The levels of NOS and MDA were determined according to the instructions of the kit (Shenzhen Zike Biotechnology).

[0064] (6) Test results

[0065] 1. Effect of combined administration of mussel polypeptide and Panax notoginseng powder on cerebral vascular permeability

[0066] The results showed that the Evans blue content of the rats in the cerebral ischemia model control group increased significantly, indicating that the model was successfully established. The test substance 1-5 groups and the positive control group were able to reduce the Evans blue content to a certain extent. The effect of the test substance 2-3 groups of mussel polypeptide and the positive control group was comparable. In the test substance 4-5 groups, the Evans blue content reduction effect after the combined administration of mussel polypeptide and Panax notoginseng was better than that of the test substance 1-3 groups administered alone. The two had the best effect and were significantly different from the normal control group. This shows that the combined use of mussel polypeptide and Panax notoginseng powder in the present invention has a synergistic effect. The results are shown in Table 2.

[0067] Table 2 Effects of Shenlongtongluo Pills on cerebral vascular permeability

[0068]

[0069] Note: vs normal control group, #, P < 0.05; vs model control group, *, P < 0.05; **, P < 0.01.

[0070] 2. Effect of combined administration of mussel polypeptide and Panax notoginseng powder on cerebral edema.

[0071] The results showed that the brain index and brain water content of the rats in the cerebral ischemia model control group increased significantly, indicating that the model was successfully established. Both the test substance 1-5 groups and the positive control group were able to reduce the brain index and water content to a certain extent, that is, they were able to reduce brain edema. Compared with the model control group, the positive control group and the test substance group were both significant. In addition, the water content of the test substance 4-5 group was lower than that of the test substance 2-3 group, indicating that the combined use of mussel polypeptide and Panax notoginseng powder in the present invention has a synergistic effect. The results are shown in Table 3.

[0072] Table 3 Effects of cerebral edema on the left and right

[0073]

[0074] Note: vs normal control group, #, P < 0.05; vs model control group, *, P < 0.05; **, P < 0.01.

[0075] 3. Effects of combined administration of mussel polypeptide and Panax notoginseng powder on NOS and MDA

[0076] The results showed that NOS and MDA increased in the brain tissue of rats in the model control group, indicating successful modeling. NOS and MDA in the brain tissue of rats in the positive control group and the test groups showed a decreasing trend, which was significant compared to the model control group, indicating that the mussel polypeptide of the present invention has a protective effect on brain damage caused by ischemia. In addition, the combined administration of mussel polypeptide and Panax notoginseng powder in test group 4-5 showed a highly significant difference compared to the model group, indicating that the combined use of mussel polypeptide and Panax notoginseng powder has a synergistic effect. The results are shown in Table 4.

[0077] Table 4 Effects on NOS and MDA

[0078]

[0079] Note: vs normal control group, #, P < 0.05; vs model control group, *, P < 0.05; **, P < 0.01.

[0080] 4. Effects of combined administration of mussel polypeptide and Panax notoginseng powder on histopathology in rats with acute incomplete cerebral ischemia

[0081] The rats in each group were killed by cervical dislocation, and the brains were removed by craniotomy. The brains were rinsed with physiological saline, dried with filter paper, and placed in a 4% paraformaldehyde solution. The tissue sections were sliced ​​and the tissue morphology was observed. Microscopic observation showed that the tissues of the rats with cerebral ischemia model had obvious pathological changes. The cytoplasm and nuclei of the nerve cells were condensed and stained darkly. The entire cell body became smaller and the number of cells decreased. The nerve cells in the brain tissue of the rats in the normal control group were normal, with clear nuclear membranes and obvious nucleoli. The nucleoli of the glial cells were clear, the cytoplasm was transparent or lightly stained, and the cell membrane and nuclear membrane were clear. The condensation and dark staining of the nerve cells in the brain tissue of the test group were significantly reduced compared with the ischemic model group; the number of cells increased, indicating that the mussel polypeptide of the present invention and its combined use with Panax notoginseng powder have a certain protective effect on the pathological changes of ischemic brain tissue in rats.

[0082] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A composition for treating cardiovascular and cerebrovascular embolic diseases, characterized in that: The composition consists of a polypeptide and Panax notoginseng; the amino acid sequence of the polypeptide is shown in SEQ ID No.

2.

2. The composition according to claim 1, characterized in that The polypeptide is derived from Mytilus edulis.

3. A pharmaceutical composition, characterized in that The composition comprises the composition according to any one of claims 1 to 2, and optionally one or more pharmaceutically acceptable carriers.

4. The pharmaceutical composition according to claim 3, characterized in that The dosage forms of the pharmaceutical composition include tablets, capsules, granules, pills, powders, suspensions, emulsions, drops, and mixtures.

5. Use of the composition according to any one of claims 1 to 2 and the pharmaceutical composition according to any one of claims 3 to 4 in the preparation of a medicament for treating cardiovascular and cerebrovascular embolic diseases, wherein the cardiovascular and cerebrovascular embolic diseases are caused by acute incomplete cerebral ischemia.

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