Natto peptide sea cucumber polysaccharide lipidosome capable of synergistically improving cardiovascular health and preparation method and application of natto peptide sea cucumber polysaccharide lipidosome

Abstract

The invention discloses a natto peptide sea cucumber polysaccharide liposome for synergistically improving cardiovascular health and a preparation method and application thereof, belongs to the field of active peptide delivery systems, and aims to solve the stability problem of natto oligopeptide in gastrointestinal digestive tracts. The phytosterol and the vitamin E are filled between phospholipid double layers of the liposome to support a gap between the phospholipid double layers, and the ratio of the phytosterol to the vitamin E is (1-3): 1, so that the natto oligopeptide liposome has the effects that the storage of the natto oligopeptide and the stability of the natto oligopeptide in gastrointestinal and digestive tracts can be improved, the bioavailability of the natto oligopeptide liposome is improved, and the bioavailability of the natto oligopeptide liposome is improved. The traditional Chinese medicine composition has synergistic effects in the aspects of anticoagulation, thrombolysis and blood fat reduction, and has a good cardiovascular protection effect.

Description

Technical Field

[0001] The present invention belongs to the field of active peptide delivery systems, and specifically relates to a preparation of natto peptide sea cucumber polysaccharide liposomes and their synergistic effect in improving cardiovascular health, especially a carrier system of sea cucumber polysaccharide-encapsulated natto peptide liposomes and its application in treating cardiovascular diseases. Background Art

[0002] Natto oligopeptides are active peptides prepared from other protein components in natto into small molecule peptides while retaining the activity of nattokinase in natto. The nattokinase contained in natto oligopeptides can directly dissolve the fibrin in the thrombus, thereby playing a role in dissolving the thrombus. It may also inhibit the formation of thrombus by affecting the aggregation function of platelets. At the same time, nattokinase can activate the fibrinolytic system in the body, promote the conversion of plasminogen into plasmin, and thus accelerate the dissolution process of thrombus. In addition, the ACE inhibitory peptides and antithrombotic peptides contained in natto oligopeptides can further provide thrombolysis and blood pressure reduction effects. At the same time, nattokinase also has the effects of lowering blood lipids and treating Alzheimer's disease.

[0003] Natto oligopeptide products can enter the blood and be used by the body only after passing through the oral-gastric-intestinal digestion and absorption system, but the digestion and absorption system environment (low pH value, etc.) and factors (pepsin, trypsin, etc.) can destroy the spatial structure of nattokinase, resulting in the reduction or loss of its thrombolytic function. Therefore, constructing an embedding delivery system for natto oligopeptides is the main way to solve the above problems. Liposomes are one of the common delivery systems, which are carrier preparations with a microcapsule structure composed of one or more concentric phospholipid bilayers. Its unique structure enables it to encapsulate water-soluble, oil-soluble and amphiphilic substances, with the hydrophilic head exposed to the aqueous phase and the hydrophobic tails gathered together to form closed vesicles with a bilayer structure. Liposomes can improve the stability of compounds, overcome cell and tissue uptake barriers, increase the biodistribution of drugs at the target site in the body, and reduce systemic toxicity. However, when liposomes are embedded in temperature changes, mechanical stirring or long-term placement, there is a tendency for physical instability to occur, and phospholipid molecules are also easily oxidized, resulting in changes in the structure and properties of liposomes. Summary of the invention

[0004] In order to solve the stability problem of the embedded material in the gastrointestinal tract, on the first aspect, according to the liposome in the embodiment of the present invention, including a microcapsule structure composed of concentric phospholipid bilayers, phytosterols and vitamin E are filled between the phospholipid bilayers of the liposome to support the gap between the phospholipid bilayers.

[0005] According to the liposome in the embodiment of the present invention, the phytosterol:vitamin E=(1-3):1 by mass.

[0006] According to the liposome in the embodiment of the present invention, the natto oligopeptide is embedded in the inner layer of the concentric phospholipid bilayer.

[0007] According to the liposome in the embodiment of the present invention, the material for forming the concentric phospholipid bilayer includes any one of soybean lecithin and Antarctic krill oil.

[0008] According to the liposome in the embodiment of the present invention, the material for forming the concentric phospholipid bilayer includes Antarctic krill oil, wherein the combination of Antarctic krill oil: the plant sterols and vitamin E = (1-5):1.

[0009] According to the liposome in the embodiment of the present invention, the material for forming the concentric phospholipid bilayer includes Antarctic krill oil, wherein the ratio of Antarctic krill oil: phytosterol: vitamin E is 6:2:1.

[0010] According to the liposome in the embodiment of the present invention, sodium alginate encapsulates the liposome.

[0011] According to the liposome in the embodiment of the present invention, sea cucumber polysaccharide encapsulates the liposome.

[0012] According to the liposome in the embodiment of the present invention, the phytosterol is any one of β-sitosterol, campesterol and stigmasterol.

[0013] According to the liposome in the embodiment of the present invention, the total phospholipid content in the Antarctic krill oil is 40% to 60%.

[0014] According to the liposome in the embodiment of the present invention, the EPA content in the Antarctic krill oil is 15% to 25%.

[0015] According to the liposome in the embodiment of the present invention, the DHA content is 7% to 15%.

[0016] According to the liposomes in the embodiments of the present invention, the astaxanthin content is ≥ 0.3 mg / g.

[0017] In a second aspect, the method for preparing liposomes according to an embodiment of the present invention comprises the following steps:

[0018] S10. Evaporating the first organic solvent dissolving the first composition into a lipid film, wherein the first composition comprises a material for forming a concentric phospholipid bilayer, phytosterols and vitamin E.

[0019] S20. Dispersing the lipid film in a second organic solvent to obtain a lipid solution.

[0020] S30. The aqueous solution containing the embedded object is added to the lipid solution, homogenized in an ice bath, and the second organic solvent is evaporated to obtain the first liposome.

[0021] The method for preparing liposomes according to the embodiment of the present invention further comprises the following steps:

[0022] S40. Extruding the first liposome and / or the second liposome through a polycarbonate membrane using a liposome extruder to obtain a second liposome.

[0023] The method for preparing liposomes according to the embodiment of the present invention further comprises the following steps:

[0024] S50. Dropping the first liposome and / or the second liposome into the stirring sea cucumber polysaccharide aqueous solution to obtain liposomes containing sea cucumber polysaccharide-wrapped embedding materials.

[0025] The method for preparing liposomes according to the embodiment of the present invention further comprises the following steps:

[0026] S50. Dropping the first liposome and / or the second liposome into the stirring sodium alginate aqueous solution to obtain liposomes with the embedded material wrapped in sodium alginate.

[0027] According to the method for preparing liposomes in an embodiment of the present invention, the ratio of phytosterol to vitamin E is (1-3):1 by mass.

[0028] According to the method for preparing liposomes in the embodiment of the present invention, the material for forming the concentric phospholipid bilayers includes any one of soybean lecithin and Antarctic krill oil.

[0029] According to the method for preparing liposomes in an embodiment of the present invention, the material for forming the concentric phospholipid bilayer includes Antarctic krill oil, wherein the combination of Antarctic krill oil: the plant sterols and vitamin E = (1-5):1.

[0030] According to the method for preparing liposomes in an embodiment of the present invention, the material for forming the concentric phospholipid bilayers includes Antarctic krill oil, wherein the ratio of Antarctic krill oil: phytosterol: vitamin E is 6:2:1.

[0031] According to the method for preparing liposomes in the embodiment of the present invention, the organic solvent is one of ethanol and chloroform.

[0032] According to the method for preparing liposomes in the embodiment of the present invention, the embedded substance is natto oligopeptide.

[0033] According to the method for preparing liposomes in the embodiment of the present invention, the natto oligopeptide is a natto peptide product containing nattokinase obtained by hydrolyzing natto with protease.

[0034] According to the method for preparing liposomes in an embodiment of the present invention, in step S10, the first organic solvent dissolving the first composition is evaporated into a lipid film, comprising: under a temperature of 40 to 50° C., the first organic solvent dissolving the first composition is evaporated under reduced pressure in a container until the first organic solvent therein is completely evaporated and the container wall is covered with the lipid film.

[0035] According to the method for preparing liposomes in the embodiment of the present invention, the reduced pressure evaporation adopts a rotary evaporator, and the container is a material holding container of the rotary evaporator.

[0036] According to the method for preparing liposomes in the embodiment of the present invention, the volume of the first organic solvent: the total mass of the second composition = (3-10): 1. The second composition includes materials for forming concentric phospholipid bilayers, phytosterols and vitamin E.

[0037] According to the method for preparing liposomes in an embodiment of the present invention, the volume of the first organic solvent: the total mass of the second composition = 5:1.

[0038] According to the method for preparing liposomes in an embodiment of the present invention, in step S20, dispersing the lipid film in a second organic solvent to obtain a lipid solution comprises:

[0039] S21. Add a second organic solvent into the container.

[0040] S22. Perform water bath ultrasound until the lipid film in the container is evenly dispersed in the second organic solvent to obtain the lipid solution.

[0041] More preferably, the volume of the second organic solvent added to the container in step S20: the total mass of the second composition = (3-10): 1, wherein the second composition includes materials for forming concentric phospholipid bilayers, phytosterols and vitamin E.

[0042] According to the method for preparing liposomes in the embodiment of the present invention, the volume of the second organic solvent: the total mass of the second composition = 5:1.

[0043] According to the method for preparing liposomes in an embodiment of the present invention, the aqueous solution with the embedded material in step S30 is added to the lipid solution, homogenized at low temperature in an ice bath, and the second organic solvent is evaporated to obtain the first liposome, comprising:

[0044] S31. The embedding material is dissolved in 5 to 20 times its volume of deionized water to obtain an embedding material aqueous solution.

[0045] S32. Add the embedding aqueous solution into the lipid solution to obtain a mixed solution.

[0046] S33. Homogenize the mixed solution in an ice bath for 1 to 2 minutes.

[0047] S34. Evaporate the second organic solvent under reduced pressure in a container at 40-50° C. to obtain the first liposome.

[0048] According to the method for preparing liposomes in an embodiment of the present invention, the mass ratio of the embedding aqueous solution to the lipid solution is (1-4):1.

[0049] According to the method for preparing liposomes in an embodiment of the present invention, the mass ratio of the embedding aqueous solution to the lipid solution is 2:1.

[0050] According to the method for preparing liposomes in the embodiment of the present invention, the low temperature range of the ice bath for ice bath homogenization is 0-8°C.

[0051] According to the method for preparing liposomes in the embodiment of the present invention, the low temperature of the ice bath for ice bath homogenization is 4°C.

[0052] According to the method for preparing liposomes in the embodiment of the present invention, the pore size of the polycarbonate membrane in step S40 is one of 50 nm, 100 nm and 200 nm.

[0053] According to the method for preparing liposomes in the embodiment of the present invention, the extrusion pressure in step S40 is 1-2 MPa.

[0054] According to the method for preparing liposomes in the embodiment of the present invention, the extrusion temperature in step S40 is 15-25°C.

[0055] According to the method for preparing liposomes in the embodiment of the present invention, the number of extrusions in step S40 is 3 to 5 times.

[0056] According to the method for preparing liposomes in the embodiment of the present invention, in step S50, the liposomes are dripped into a stirred sea cucumber polysaccharide aqueous solution to obtain liposomes with sea cucumber polysaccharide-encapsulated embedding materials, including:

[0057] S51. Slowly and uniformly drop the second liposome into the stirring 0.1% to 1.0% sea cucumber polysaccharide aqueous solution.

[0058] S52. Stir for 5 to 10 minutes to obtain liposomes containing sea cucumber polysaccharide-encapsulated materials.

[0059] According to the method for preparing liposomes in an embodiment of the present invention, the sea cucumber polysaccharide is a polysaccharide mixture extracted from sea cucumbers and containing sea cucumber chondroitin sulfate and sea cucumber fucoidan sulfate.

[0060] According to the liposome preparation method in the embodiment of the present invention, the total content of sea cucumber chondroitin sulfate and sea cucumber fucoidan sulfate is 80% to 95%.

[0061] According to the method for preparing liposomes in an embodiment of the present invention, the mass ratio of the sea cucumber polysaccharide aqueous solution to the second liposome is (1-4):1.

[0062] According to the method for preparing liposomes in the embodiment of the present invention, the stirring speed is 200-500 r / min.

[0063] On the third aspect, the liposome is prepared according to any preparation method described in the second aspect of the present invention.

[0064] In a fourth aspect, use of the liposome according to any one of the first aspect and the second aspect of the present invention in the preparation of a method for preventing and / or treating cardiovascular diseases.

[0065] Beneficial effects:

[0066] In the first aspect, a certain amount of cholesterol is usually added during the preparation process to improve the stability of liposomes. It can be inserted into the phospholipid bilayer, increase the rigidity and stability of the membrane, reduce the fluidity of the membrane, and thus reduce the leakage of drugs in liposomes. Excessive intake of cholesterol can easily cause diseases such as atherosclerosis and cancer, so there is research on the use of plant sterols to replace cholesterol. However, high concentrations of plant mixed sterols have a greater impact on the gastrointestinal digestion stability of liposomes, the leakage rate of the encapsulated contents is higher, and its ability to reduce the fluidity of the phospholipid bilayer is lower than that of cholesterol, affecting the stability of liposomes. The present invention finds that plant sterols and vitamin E are mixed according to the ratio of the present invention, which can better replace the role of cholesterol in liposomes, and compared with the preparation of liposomes using plant sterols alone, it can better provide a filling effect between phospholipid molecules, so that plant sterols and vitamin E are filled between the phospholipid bilayers of liposomes to support the gap between the phospholipid bilayers, thereby reducing fluidity, improving the rigidity stability of liposomes, reducing deformability, aggregation, and enhancing the buffering of collisions between liposomes.

[0067] On the second aspect, if the phospholipid, phytosterol and vitamin E mixture is directly mixed with the aqueous phase to form liposomes, some phytosterols or vitamin E will be embedded in the closed embedding space of the phospholipid bilayer structure, reducing the embedding effect of phytosterols and vitamin E on the gap between phospholipid molecules, resulting in poor structural rigidity stability. For this reason, the present invention embeds phytosterols and vitamin E between phospholipid molecules during the formation of lipid membrane. When the subsequent phospholipid molecules are dispersed and combined to form liposomes, the gap between phospholipid molecules is utilized to have a certain locking effect on phytosterols and vitamin E. When phospholipids are formed into liposomes, especially when the closed embedding space of the phospholipid bilayer structure is formed by phospholipid molecules, it is possible to avoid embedding phytosterols and vitamin E, so that phytosterols and vitamin E can be more between the phospholipid bilayers for support, improve structural rigidity stability, reduce deformability and enhance the buffering of collisions between liposomes.

[0068] On the third side, phospholipids are easily oxidized, which leads to the destruction of liposome membranes. The Antarctic krill oil used in the present invention contains astaxanthin, a natural strong antioxidant, and by embedding a certain amount of vitamin E between phospholipid molecules, the antioxidant capacity of the formed liposomes is greatly improved, and the chemical stability of the structure is improved because it is not easily oxidized.

[0069] In the fourth aspect, during the preparation process, the aqueous phase and the lipid solution are mixed and then homogenized at low temperature and then extruded by a liposome extruder, so that the particle size distribution of the formed liposomes is narrow and the stability is good. Finally, the blocking effect formed by the coating of the sea cucumber polysaccharide molecules on the surface of the liposomes resists the aggregation between the liposomes and reduces the leakage of the encapsulated natto oligopeptide, thereby further improving the stability of the liposomes.

[0070] In the fifth aspect, the present invention has the effect of increasing the particle size of liposomes by embedding phytosterols and vitamin E between phospholipid molecules and coating sea cucumber polysaccharide molecules, thereby improving the encapsulation rate of natto oligopeptides.

[0071] In the sixth aspect, the sea cucumber polysaccharide-encapsulated natto oligopeptide liposomes of the present invention can better resist the low pH environment and the destruction of the natto oligopeptide structure by digestive enzymes in the gastrointestinal tract, thereby improving its stability, cell permeability and bioavailability.

[0072] In the seventh aspect, after the liposomes of the present invention are absorbed into the blood in the intestine, the three main components of the carrier system, namely, natto oligopeptides used as core materials, wall materials and coatings to improve cardiovascular health, Antarctic krill oil used as phospholipid materials to construct a phospholipid bilayer structure, and sea cucumber polysaccharides that resist aggregation between liposomes and reduce leakage of encapsulated natto oligopeptides, all have the effect of improving cardiovascular health, and the mechanism and specific effects are also different. The delivery system of the present invention not only has stable delivery coordination and efficacy, but also takes into account the auxiliary and coordinated natto oligopeptides in medicinal efficacy as a delivery structure, improving the complementarity and comprehensiveness of treatment. Among them, the two long-chain fatty acids of phospholipids in Antarctic krill oil are mostly composed of EPA and DHA, and these two unsaturated fatty acids have been proven to have certain effects of reducing triglycerides, increasing high-density lipoprotein cholesterol, and reducing low-density lipoprotein cholesterol. At the same time, it also has the effects of inhibiting platelet aggregation, reducing blood viscosity, reducing inflammatory cell infiltration in vascular plaques, and regulating lipid metabolism in plaques. Astaxanthin contained in Antarctic krill oil is also a powerful antioxidant and anti-inflammatory agent, reducing oxidative stress and inflammatory response of blood vessel lining cells. Natto oligopeptides inhibit the formation of blood clots through nattokinase and antithrombotic peptide components, and ACE inhibitory peptide components dilate blood vessels, improve blood flow pressure, and reduce endothelial damage and inflammation. Sea cucumber polysaccharides also have cardiovascular maintenance activities including regulating blood lipids, anti-thrombosis and protecting endothelial cells. The synergistic effect of the three has a more significant effect on improving cardiovascular health than taking them alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Fluorescence polarization plot of liposomes.

[0074] Figure 2 Graph of TBARS change rate of liposomes.

[0075] Figure 3 Plot of liposome leakage rate. DETAILED DESCRIPTION

[0076] The present invention is further described below by way of examples. It should be noted that the examples do not limit the scope of protection claimed for the present invention.

[0077] The present invention provides a natto peptide sea cucumber polysaccharide liposome delivery system, and the preparation method is as follows:

[0078] S1. Fully dissolve Antarctic krill oil, plant sterols and vitamin E in an organic solvent, evaporate under reduced pressure at 40-50°C until the organic solvent is completely evaporated, so that a lipid film is formed in the container.

[0079] Furthermore, the total phospholipid content of the Antarctic krill oil is 40% to 60%, the EPA content is 15% to 25%, the DHA content is 7% to 15%, and the astaxanthin content is ≥ 0.3 mg / g;

[0080] Further, the phytosterol is one of β-sitosterol, campesterol, and stigmasterol, preferably β-sitosterol;

[0081] Furthermore, the organic solvent is one of ethanol and chloroform;

[0082] Furthermore, the volume of the organic solvent is 3 to 10 times the total mass of the Antarctic krill oil, phytosterols and vitamin E, preferably 5 times.

[0083] Furthermore, the mass ratio of the Antarctic krill oil, phytosterols and vitamin E is Antarctic krill oil: phytosterols + vitamin E = 1 to 5:1, wherein phytosterols: vitamin E = 1 to 3:1, preferably Antarctic krill oil: phytosterols: vitamin E = 6:2:1

[0084] S2. Add the same volume of organic solvent as in S1 into the container and perform water bath sonication until the lipid film is evenly dispersed in the solvent as the lipid solution.

[0085] S3. Dissolve natto oligopeptide in 5 to 20 times the volume of deionized water to prepare the aqueous phase, add the natto oligopeptide aqueous solution to the lipid solution in S2, homogenize at low temperature in an ice bath for 1 to 2 minutes, and evaporate under reduced pressure at 50°C to remove the organic solvent.

[0086] Furthermore, the natto oligopeptide refers to a natto peptide product containing nattokinase obtained by subjecting natto to protease hydrolysis.

[0087] Furthermore, the mass ratio of the natto oligopeptide aqueous solution to the lipid solution is 1 to 4:1, preferably 2:1.

[0088] Furthermore, the natto oligopeptide may be the natto oligopeptide in the applicant's authorized patent CN118325994B.

[0089] S4. Extrude through a polycarbonate membrane using a liposome extruder to obtain natto oligopeptide liposomes (NP-L).

[0090] Furthermore, the pore size of the polycarbonate membrane is one of 50 nm, 100 nm, and 200 nm; the extrusion pressure is 1 to 2 MPa; the extrusion temperature is 15 to 25° C.; and the number of extrusions is 3 to 5 times.

[0091] S5. Weigh sea cucumber polysaccharides to prepare 0.1% to 1.0% sea cucumber polysaccharide aqueous solution, slowly and evenly drop the natto oligopeptide liposomes in S4 into the stirring sea cucumber polysaccharide aqueous solution, and stir for 5 to 10 minutes to obtain sea cucumber polysaccharide-encapsulated natto oligopeptide liposomes (SCP-NP-L).

[0092] Furthermore, the sea cucumber polysaccharide is a polysaccharide mixture containing sea cucumber chondroitin sulfate and sea cucumber fucoidan sulfate extracted from sea cucumber, and the total content of sea cucumber chondroitin sulfate and sea cucumber fucoidan sulfate is 80% to 95%;

[0093] Furthermore, the mass ratio of the sea cucumber polysaccharide aqueous solution to the liposome is 1 to 4:1.

[0094] Furthermore, the stirring speed is 200-500 r / min.

[0095] Furthermore, the container is a rotary evaporation apparatus, and rotary evaporation is beneficial to embedding plant sterols and vitamin E into phospholipid molecules during the lipid film preparation stage.

[0096] Among them, studies have reported that natto oligopeptides inhibit the formation of thrombus through nattokinase and antithrombotic peptide components, and ACE inhibitory peptide components dilate blood vessels, improve blood flow pressure, and reduce the occurrence of vascular endothelial damage and inflammation. Antarctic krill oil is extracted from Antarctic krill and is mainly divided into two types: triglyceride krill oil without phospholipids and phospholipid krill oil rich in phospholipids. The phospholipid-type Omega-3 fatty acids in Antarctic krill oil have EPA and DHA as hydrophobic ends at both ends of the glycerol structure, and choline and phosphate esters as hydrophilic ends. They have a strong emulsification type. After being absorbed into the blood by the small intestine, they can make the cholesterol particles on the blood vessel wall smaller and keep them in a suspended state, so that they can be used by tissues and organs through the blood vessel wall, thereby reducing the cholesterol content in the blood vessels. At the same time, EPA and DHA can reduce the synthesis of triglycerides and cholesterol in the human body, and play a role in lowering blood lipids. In addition, studies have reported the anti-thrombotic effect of Antarctic krill oil, which prevents the formation of blood clots by inhibiting endothelial dysfunction, regulating coagulation, and reducing inflammation. The effect is similar to that of the commonly used drug aspirin. Therefore, Antarctic krill oil has a good effect on cardiovascular health. Sea cucumber polysaccharides mainly include fucosylated chondroitin sulfate (FCS) and fucoidan sulfate (FUC), which are natural high-molecular anionic polysaccharides. It has good biocompatibility and degradability. Studies have reported that sea cucumber polysaccharides reduce the risk of atherosclerotic plaque formation by lowering blood lipid levels, inhibiting platelet aggregation, and protecting vascular endothelial cells. The synergistic effect of the three has a more significant effect on improving cardiovascular health than taking them alone. For example, for atherosclerotic plaques, on the one hand, it alleviates the formation of atherosclerotic plaques by reducing the accumulation of fat in the inner wall of blood vessels and the inflammatory response; on the other hand, when plaques have already occurred, it reduces the serious thrombosis that blocks blood vessels when the plaques are damaged, accelerates the healing of plaques after rupture or erosion, and prevents infarction caused by thrombus detachment; regulates the lipid transport metabolism inside and outside the plaque, and alleviates the further thickening and enlargement of the plaque.

[0097] The carrier system of the sea cucumber polysaccharide-encapsulated natto peptide liposomes of the present invention can solve the problem that the active ingredients of natto are sensitive to strong acid, alkali and high temperature environments. The system can improve the storage of natto oligopeptides and their stability in the gastrointestinal digestive tract, and improve their bioavailability. At the same time, the natto oligopeptides, Antarctic krill oil and sea cucumber polysaccharides in the system, after being absorbed, respectively act synergistically in anticoagulation, thrombolysis and lowering blood lipids, and have a good cardiovascular protective effect.

[0098] Example 1: Preparation of SCP-NP-L1, comprising the following steps:

[0099] S1. Weigh 30g of Antarctic krill oil, 10g of β-sitosterol and 5g of vitamin E respectively and fully dissolve them in 225ml of ethanol, evaporate under reduced pressure at 50°C until the organic solvent evaporates completely, so that a lipid film is formed in the container. The total phospholipid content in Antarctic krill oil is 60%, the EPA content is 20%, the DHA content is 10%, and the astaxanthin content is 0.3mg / g.

[0100] S2. Add 225 ml of ethanol into the container and perform water bath sonication for 1 min until the lipid film is evenly dispersed in the solvent as the lipid solution.

[0101] S3. Prepare natto oligopeptide. Dissolve 10 g of natto oligopeptide powder in 10 times the volume of deionized water to prepare the aqueous phase. Add 5 g of the lipid solution in S2 to the natto oligopeptide aqueous solution. Homogenize at low temperature in an ice bath at 4°C for 1 min. Evaporate at 50°C to remove ethanol.

[0102] S4. The liposome was extruded through a 200 nm polycarbonate membrane using a liposome extruder, with a base pressure of 1 MPa, an extrusion temperature of 25°C, and 5 extrusions to obtain 13.5 g of natto oligopeptide liposomes (NP-L1).

[0103] S5. Weigh 0.1 g of sea cucumber polysaccharide with a purity of 90% to prepare a 0.2% sea cucumber polysaccharide aqueous solution, take 5 g of natto oligopeptide liposomes and slowly and uniformly drip them into the stirring sea cucumber polysaccharide aqueous solution, stir at 300 r / min for 10 min to obtain sea cucumber polysaccharide-encapsulated natto oligopeptide liposomes (SCP-NP-L1).

[0104] Wherein, the preparation of natto oligopeptide comprises:

[0105] S31. Take 100 g of natto freeze-dried powder and dissolve it in 1000 ml of physiological saline, and take the permeate through a 29,000 molecular weight ultrafiltration membrane to obtain components with a molecular weight lower than 29,000.

[0106] S2. The permeate is passed through a 26,000 molecular weight ultrafiltration membrane to obtain a retentate, and the component with a molecular weight higher than 26,000 but lower than 29,000 is the nattokinase component F1. The first ultrafiltration retentate and the second ultrafiltration permeate are combined to form the natto component F2.

[0107] S33. The natto component F2 was hydrolyzed under the following conditions: trypsin: chymotrypsin = 1:1-3, enzyme addition amount: 1-3%, hydrolysis temperature: 37-45°C, hydrolysis pH: 7.5-8.5, and hydrolysis time of 3-6 hours to obtain a natto hydrolyzate.

[0108] S34. The second enzymatic hydrolysate was separated by a nanofiltration membrane with a cutoff of 2000Da, and the permeate was collected and eluted using a SP Sepharose High Performance strong cation exchange column with a concentration gradient of 0-40min:0-1M NaCl, and the eluate with a retention time of 11-19min was collected as the natto enzymatic hydrolysate component F3. The natto enzymatic hydrolysate component F4 and the nattokinase component F1 were fully mixed and freeze-dried to obtain natto oligopeptide powder.

[0109] Example 2: Preparation of SCP-NP-L2: The preparation steps of SCP-NP-L2 are the same as those in Example 1, except that 30 g of Antarctic krill oil, 10 g of β-sitosterol and 5 g of vitamin E in S1 are replaced with 30 g of Antarctic krill oil and 15 g of β-sitosterol to prepare the lipid membrane, and natto oligopeptide liposomes (NP-L2) are obtained. Natto oligopeptide liposomes (SCP-NP-L2) are encapsulated with sea cucumber polysaccharides.

[0110] Example 3: Preparation of SCP-NP-L3: The preparation steps of SCP-NP-L3 are the same as those in Example 1, except that 30 g of Antarctic krill oil is replaced with 30 g of soybean lecithin in S1 to obtain natto oligopeptide liposomes (NP-L3), and sea cucumber polysaccharides encapsulate natto oligopeptide liposomes (SCP-NP-L3).

[0111] Example 4: Preparation of SCP-NP-L4: The preparation steps of SCP-NP-L4 are the same as those of Example 1, except that the lipid membrane preparation step of S1 is omitted. 30 g of Antarctic krill oil, 10 g of β-sitosterol and 5 g of vitamin E are added to 225 ml of ethanol and mixed, and the mixture is ultrasonicated in a water bath for 1 min as a lipid solution. Natto oligopeptide liposomes (NP-L4) are obtained, and sea cucumber polysaccharides encapsulate natto oligopeptide liposomes (SCP-NP-L4).

[0112] Example 5: Preparation of SA-NP-L: Sodium alginate encapsulated natto oligopeptide liposomes The preparation steps of SA-NP-L were the same as those in Example 1, except that sodium alginate SA was used in S5 to replace sea cucumber polysaccharide to prepare SA-NP-L.

[0113] Experimental description:

[0114] (1) Liposome phospholipid membrane fluidity experiment: The DPH fluorescent probe was used to measure the fluidity of the liposome membrane. In a membrane environment with high fluidity, the fluorescent probe rotates quickly and has a small polarization degree; in a membrane environment with low fluidity (high rigidity), the fluorescent probe rotates slowly and has a large polarization degree. The liposome suspension was mixed evenly with 500 μL DPH tetrahydrofuran solution (0.086 mmol / L), and the mixed solution was incubated at 37°C for 1 hour. The fluorescence polarization degree of the DPH probe was measured using a multifunctional microplate reader. The parameters of the microplate reader were set as follows: excitation wavelength 360 nm, emission wavelength 430 nm. The fluorescence polarization degree FP was calculated according to the following formula: FP = I0-G*I 90 / I0+G*I 90 Where: I0 and I 90 are the fluorescence intensities of the fluorescent probes in the horizontal and vertical channels, respectively; G is the correction factor of the horizontal or vertical channel.

[0115] (2) Anti-peroxidation ability test of liposomes: The polyunsaturated fatty acid chains in the phospholipid molecular skeleton easily cause lipid peroxidation of liposomes during processing or storage, resulting in structural instability and leakage of encapsulated substances. The anti-peroxidation ability of liposomes was determined by the thiobarbituric acid reactant (TBARS) method. Prepare trichloroacetic acid-thiobarbituric acid-hydrochloric acid (TBA-TCA-HCl) solution, place the liposomes in a 25℃ water bath with the lid open and incubate for 24 hours. Take samples every 4 hours and add 5mL of TBA-TCA-HCl solution. After boiling in a water bath for 30 minutes, take out and cool in an ice bath. The fluorescence intensity of TBARS is measured by a fluorescence spectrophotometer. The TBARS change rate is calculated as the ratio of the increase in TBARS fluorescence intensity in the sample at time t relative to the 0h sample to the TBARS fluorescence intensity in the sample at 0h.

[0116] (3) Determination of particle size, encapsulation efficiency and storage stability of liposomes: A laser particle size analyzer was used to determine the particle size of the liposomes in each example. Take 1g of liposomes in a centrifuge tube, add twice the volume of ultrapure water, centrifuge at 4°C and 5000r / min for 30 minutes, and take the supernatant. The BCA method was used to determine the concentration of free natto oligopeptides (C1) and the total natto oligopeptides (C2) in the supernatant at 562nm. The encapsulation efficiency of liposomes was calculated using the formula EE (%) = (C2-C1) / C2*100. The retention rate after 50 days at 4°C in a dark room was calculated using the formula retention rate (%) = EE t Calculated using / EE0*100.

[0117] (4) Determination of in vitro digestion stability of liposomes: 2 g of liposomes and natto oligopeptide powder were diluted with 4 times deionized water, and 10 ml of simulated oral digestive fluid (SSF) containing α-amylase (75 U / ml) and calcium chloride (1.5 mM) was added, mixed thoroughly, and oscillated in a water bath at 37°C and pH 7.0 for 10 min in the dark. After digestion, 10 ml of simulated gastric digestive fluid (SGF) containing pepsin (60 U / ml) and calcium chloride (0.15 mM) was added, mixed thoroughly, and incubated at 37°C and pH 3.0 for 10 min. The mixture was shaken in a water bath and protected from light for 2 hours. After digestion, 10 ml of simulated small intestinal digestive fluid (SIF) containing trypsin (100 U / ml), bile salt (10 mM) and calcium chloride (0.6 mM) was added and mixed thoroughly. The mixture was shaken in a water bath at 37°C and pH 7.0 and protected from light for 2 hours. After digestion, the mixture was centrifuged at 4°C and 5000 r / min for 30 minutes to obtain the supernatant. The leakage rate of natto oligopeptide was determined. The leakage rate was calculated according to the formula: leakage rate (%) = 100-retention rate. The retention rate was obtained according to the method described in Experimental Example 1.

[0118] (5) Liposomes for ApoE - / - Experiment on the effect of ApoE on the formation of atherosclerotic plaque in mice: 60 ApoE - / - Mice were randomly divided into model group, NP group, NP-L1 group, SCP-NP-L1 group, SCP-NP-L3 group, and SA-NP-L group, with 10 mice in each group. After 1 week of adaptive feeding, they were all fed with high-fat diet [1.25% cholesterol (TC) + 15% triglyceride (TG)]. 10 C57BL / 6 mice were used as the control group and fed with ordinary diet. While feeding high-fat diet, each intervention group was gavaged with 10 mg / (kg·d) natto oligopeptide powder / liposome, and the control group and model group were gavaged with the same amount of normal saline for 12 weeks. After the end of the administration, the mice were anesthetized with 1% sodium pentobarbital, and 2 ml of blood was collected from the carotid artery, left to stand for 30 minutes, and centrifuged at 4℃ and 3000r / min for 15 minutes. The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in the supernatant were detected by automatic biochemical analyzer. Five mice carotid aortas were randomly selected from each group and fixed with 10% formaldehyde at 4°C for 24 h, embedded in paraffin, and stained with HE to observe the formation of aortic plaques in mice. Images were analyzed with Image Pro Plus 6.0 software, and the plaque area was calculated. Plaque area ratio (%) = plaque area / aortic inner wall cross-sectional area × 100%.

[0119] Experimental Example 1. The results of the phospholipid membrane fluidity test of liposomes are as follows Figure 1As shown in the figure, NP-L1, which was embedded in the lipid bilayer using plant sterols and vitamin E and processed by lipid membrane preparation, had the highest fluorescence polarization degree (P < 0.05), that is, higher lipid layer rigidity, and NP-L2, which only used plant sterols, had the lowest fluorescence polarization degree (P < 0.05). Taking β-sitosterol as an example, the molecular length of plant sterols is about 1-2nm, and the thickness of the lipid bilayer membrane is about 2.5-3nm. Therefore, β-sitosterol is similar to cholesterol and is anchored to the lipid bilayer on one side. The length of vitamin E molecules is close to that of the lipid bilayer membrane, and it is anchored to the lipid bilayer on both sides, which can provide higher rigidity. NP-L3 using soybean lecithin also showed lower rigidity than NP-L1 (P < 0.05), which is speculated to be caused by structural differences such as the saturation and chain length of the fatty acid chains of the phospholipid components in krill oil and soybean lecithin, and the ratio of lecithin / sphingomyelin. NP-L4 that has not undergone lipid membrane preparation also exhibits lower rigidity than NP-L1 (P < 0.05), indicating that the lipid membrane preparation step (adding phytosterols and vitamin E to embed into the lipid bilayer before the phospholipid molecules form a closed embedding space of the phospholipid bilayer structure) helps to load phytosterols and vitamin E into the lipid bilayer and improve the rigidity of the lipid layer.

[0120] Experimental Example 2. The results of the liposome anti-peroxidation test are as follows Figure 2 As shown, NP-L1 has the highest antioxidant capacity, followed by NP-L2 without vitamin E, and NP-L3 with soybean lecithin has the weakest antioxidant capacity (P < 0.05). The present invention utilizes the unsaturated fatty acid chains rich in Antarctic krill oil, astaxanthin with strong antioxidant properties, and vitamin E embedded in the lipid layer structure to provide liposomes with antioxidant capacity.

[0121] Experimental Example 3. The results of the particle size, encapsulation efficiency and storage stability of liposomes are shown in Table 1:

[0122] Table 1

[0123] Example Particle size (nm) Encapsulation rate (%) Retention rate (%) NP-L1 158.7±15.3 <![CDATA[68.25±0.81 a ]]> <![CDATA[89.31±0.44 a ]]> NP-L2 137.2±29.0 <![CDATA[62.10±0.93 c ]]> <![CDATA[77.10±1.61 b ]]> NP-L3 160.6±10.1 <![CDATA[67.84±0.50 a ]]> <![CDATA[82.26±0.74 c ]]> NP-L4 134.4±31.0 <![CDATA[61.92±1.38 c ]]> <![CDATA[84.29±1.15 c ]]> SCP-NP-L1 210.2±12.5 <![CDATA[75.21±0.60 b ]]> <![CDATA[95.60±0.38 d ]]> SCP-NP-L2 193.7±11.3 <![CDATA[67.34±1.19 a ]]> <![CDATA[83.04±0.90 c ]]> SCP-NP-L3 207.0±10.6 <![CDATA[73.83±1.22 b ]]> <![CDATA[86.94±1.25 e ]]> SCP-NP-L4 183.3±35.9 <![CDATA[65.88±1.64 a ]]> <![CDATA[95.33±0.87 d ]]> SA-NP-L 201.1±20.2 <![CDATA[74.09±1.35 b ]]> <![CDATA[94.11±0.52 d ]]>

[0124] Note: Samples with different letters in the same column have significant differences within the group, P < 0.05

[0125] As can be seen from Table 1, the liposome sample coated with sea cucumber polysaccharide has a significant improvement in encapsulation efficiency and retention rate compared with the natto oligopeptide liposome NP-L in the corresponding example (P < 0.05); there is no significant difference in the encapsulation efficiency between SCP-NP-L1 and SCP-NP-L3 prepared with soybean lecithin and SA-NP-L prepared with sodium alginate (P > 0.05), but the retention rate of SCP-NP-L1 is significantly higher than that of SCP-NP-L3 (P < 0.05). It is speculated that the antioxidant components in Antarctic krill oil have a promoting effect on maintaining the stability of liposomes, and SCP-NP-L -NP-L4 and SA-NP-L have the same storage stability as SCP-NP-L1 (P>0.05); the encapsulation efficiency of NP-L2, which only uses β-sitosterol to provide phospholipid layer rigidity, and NP-L4 that has not been treated with lipid membrane is significantly lower than that of NP-L1 (P<0.05). The rigidity provided by phytosterols alone when used to prepare liposomes is insufficient, which is improved after being used in combination with vitamin E. The particle size distribution and encapsulation efficiency of NP-L4 indicate that the S1 lipid membrane step in Example 1 helps to improve the embedding of phytosterols and vitamin E in the lipid layer and the uniformity of particle size, thereby improving the encapsulation efficiency.

[0126] Experimental Example 4. Results of in vitro digestion stability test of liposomes Figure 3 As shown in the results, the degradation rate of unencapsulated natto oligopeptides after simulated digestion was close to 60%, and the leakage rates of the sample groups encapsulated by liposomes decreased significantly to varying degrees (P < 0.05). Only the leakage rate of NP-L1 encapsulated by liposomes decreased to 35.34%, and the leakage rates of SCP-NP-L1, SCP-NP-L2, SCP-NP-L3, SCP-NP-L4, and SA-NP-L in the sample groups encapsulated by polysaccharides further decreased (P < 0.05). Among them, due to the poor stability of the liposome structure, the leakage rates of SCP-NP-L2 and SCP-NP-L4 groups after simulated digestion were higher than those of the other three groups even after being encapsulated by sea cucumber polysaccharides (P < 0.05).

[0127] Experimental Example 5. Liposomes and ApoE - / - The results of blood lipid determination and plaque area ratio in the experiment on the formation of atherosclerotic plaques in mice are shown in Table 2:

[0128] Table 2

[0129]

[0130] Note: Samples with different letters in the same column have significant differences within the group, P < 0.05

[0131] As shown in Table 2, the SCP-NP-L1 group significantly reduced the total cholesterol, triglyceride and low-density lipoprotein content in the model group, increased the high-density lipoprotein content, and inhibited the formation of plaques in the aorta, which had the best improvement effect compared with other experimental groups (P < 0.05). Although the SA-NP-L group encapsulated with sodium alginate was at the same level as the SCP-NP-L1 group in terms of encapsulation rate and stability, it did not have the activity of sea cucumber polysaccharides in regulating blood lipids and anti-thrombosis, and the improvement effect was relatively inferior under the same dose intervention. The SCP-NP-L3 group encapsulated with soybean lecithin had a certain degree of decline in stability in long-term storage and simulated gastrointestinal digestion compared with the SCP-NP-L1 group using Antarctic krill oil, thereby affecting the intervention effect of mouse atherosclerotic plaques.

[0132] In summary, the sea cucumber polysaccharide-encapsulated natto oligopeptide liposomes provided by the present invention effectively improve the stability and bioavailability of natto oligopeptides in the gastrointestinal environment, have high biocompatibility, long blood circulation time, and have a significant improvement effect on cardiovascular health.

[0133] Finally, it should be noted that the above examples are only some specific embodiments of the present invention, and all derivatives that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A liposome comprising a microcapsule structure composed of a concentric phospholipid bilayer, characterized in that: Phytosterols and vitamin E fill between the phospholipid bilayers of the liposome to support the gaps between the phospholipid bilayers.

2. The liposome according to claim 1, characterized in that in, By mass, the phytosterol:vitamin E=(1-3):

1.

3. The liposome according to any one of claims 1 to 2, characterized in that Preferably, the natto oligopeptide is embedded in the inner layer of the concentric phospholipid bilayer; Preferably, the material for forming the concentric phospholipid bilayers includes any one of soybean lecithin and Antarctic krill oil; Preferably, the material for forming the concentric phospholipid bilayer comprises Antarctic krill oil, wherein the combination of Antarctic krill oil: the plant sterol and vitamin E = (1-5): 1; Preferably, the material for forming the concentric phospholipid bilayers includes Antarctic krill oil, wherein the ratio of Antarctic krill oil: phytosterol: vitamin E is 6:2:

1.

4. The liposome according to any one of claims 1 to 3, characterized in that Preferably, sodium alginate encapsulates the liposomes; Preferably, sea cucumber polysaccharide encapsulates the liposome.

5. The liposome according to any one of claims 1 to 4, characterized in that Preferably, the phytosterol is any one of β-sitosterol, campesterol, and stigmasterol; Preferably, the total phospholipid content in the Antarctic krill oil is 40% to 60%; More preferably, the EPA content in the Antarctic krill oil is 15% to 25%; More preferably, the DHA content is 7% to 15%; More preferably, the astaxanthin content is ≥ 0.3 mg / g.

6. A method for preparing liposomes, characterized in that: The steps include: S10. Evaporating the first organic solvent dissolving the first composition into a lipid film; wherein the first composition comprises a material for forming a concentric phospholipid bilayer, phytosterols and vitamin E; S20. dispersing the lipid film in a second organic solvent to obtain a lipid solution; S30. The aqueous solution having the embedded material is added to the lipid solution, homogenized in an ice bath, and the second organic solvent is evaporated to obtain a first liposome; Preferably, the method further comprises the steps of: S40. Extruding the first liposome and / or the second liposome through a polycarbonate membrane from a liposome extruder to obtain a second liposome; More preferably, the method further comprises the steps of: S50. dropping the first liposome and / or the second liposome into the stirred sea cucumber polysaccharide aqueous solution to obtain liposomes with sea cucumber polysaccharide-encapsulated embedding material; More preferably, the method further comprises the steps of: S50. Dropping the first liposome and / or the second liposome into the stirring sodium alginate aqueous solution to obtain liposomes with the embedded material wrapped in sodium alginate.

7. The preparation method according to claim 6, characterized in that: Preferably, by mass, the phytosterol: vitamin E = (1-3): 1; Preferably, the material for forming the concentric phospholipid bilayers includes any one of soybean lecithin and Antarctic krill oil; Preferably, the material for forming the concentric phospholipid bilayer includes Antarctic krill oil, wherein the combination of Antarctic krill oil: the plant sterol and vitamin E = (1-5): 1; Preferably, the material for forming the concentric phospholipid bilayer includes Antarctic krill oil, wherein the ratio of Antarctic krill oil: phytosterol: vitamin E is 6:2:1; Preferably, the organic solvent is one of ethanol and chloroform; Preferably, the embedded substance is natto oligopeptide; More preferably, the natto oligopeptide is a natto peptide product containing nattokinase obtained by protease hydrolysis of natto; Preferably, in step S10, evaporating the first organic solvent dissolving the first composition into a lipid film comprises: evaporating the first organic solvent dissolving the first composition under reduced pressure in a container at a temperature of 40 to 50° C. until the first organic solvent therein is completely evaporated and the container wall is covered with the lipid film; More preferably, the reduced pressure evaporation adopts a rotary evaporation apparatus, and the container is a material holding container of the rotary evaporation apparatus; Preferably, the volume of the first organic solvent: the total mass of the second composition = (3-10): 1; wherein the second composition comprises a material for forming a concentric phospholipid bilayer, phytosterols and vitamin E; More preferably, the volume of the first organic solvent: the total mass of the second composition = 5:

1.

8. The preparation method according to any one of claims 6 to 7, characterized in that: Preferably, in step S20, dispersing the lipid film in a second organic solvent to obtain a lipid solution comprises: S21. Adding a second organic solvent into the container; S22. water bath sonication until the lipid film in the container is uniformly dispersed in the second organic solvent to obtain the lipid solution; More preferably, the volume of the second organic solvent added to the container in step S20: the total mass of the second composition = (3-10): 1, wherein the second composition comprises a material for forming a concentric phospholipid bilayer, phytosterols and vitamin E; More preferably, the volume of the second organic solvent: the total mass of the second composition = 5:

1.

9. The preparation method according to any one of claims 6 to 8, characterized in that: Preferably, the aqueous solution with the embedded material in step S30 is added to the lipid solution, homogenized at low temperature in an ice bath, and the second organic solvent is evaporated to obtain the first liposome, comprising S31. The embedded material is dissolved in 5 to 20 times its volume of deionized water to obtain an embedded material aqueous solution; S32. adding the embedding aqueous solution to the lipid solution to obtain a mixed solution; S33. Homogenize the mixture in an ice bath for 1 to 2 minutes; S34. Evaporating the second organic solvent under reduced pressure in a container at 40 to 50° C. to obtain the first liposome; Preferably, the mass ratio of the embedding aqueous solution to the lipid solution is (1-4):1; More preferably, the mass ratio of the embedding aqueous solution to the lipid solution is 2:1; More preferably, the low temperature range of the ice bath for ice bath homogenization is 0 to 8°C; More preferably, the ice bath low temperature of ice bath homogenization is 4°C Preferably, in step S40, the pore size of the polycarbonate membrane is one of 50 nm, 100 nm, and 200 nm; Preferably, the extrusion pressure in step S40 is 1-2 MPa; Preferably, the extrusion temperature in step S40 is 15 to 25°C; Preferably, the number of extrusions in step S40 is 3 to 5 times.

10. The preparation method according to any one of claims 6 to 9, characterized in that: In step S50, the liposomes are dropped into the stirred sea cucumber polysaccharide aqueous solution to obtain the liposomes with sea cucumber polysaccharide-encapsulated embedding material, including S51. slowly and uniformly dripping the second liposome into the stirring 0.1% to 1.0% sea cucumber polysaccharide aqueous solution; S52. Stir for 5 to 10 minutes to obtain liposomes containing the sea cucumber polysaccharide-encapsulated material; Preferably, the sea cucumber polysaccharide is a polysaccharide mixture extracted from sea cucumbers containing sea cucumber chondroitin sulfate and sea cucumber fucoidan sulfate; More preferably, the total content of sea cucumber chondroitin sulfate and sea cucumber fucoidan sulfate is 80% to 95%; Preferably, the mass ratio of the sea cucumber polysaccharide aqueous solution to the second liposome is (1-4):1; Preferably, the stirring speed is 200-500 r / min.

11. Liposome prepared by the preparation method according to any one of claims 6 to 10.

12. Use of the liposome according to any one of claims 1-5 and 11 in the preparation of a drug for preventing and / or treating cardiovascular diseases.

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