Sea cucumber polysaccharide, natto and oligopeptide composition capable of efficiently lowering lipid, reducing plaque and inhibiting thrombus and application of sea cucumber polysaccharide, natto and oligopeptide composition

Abstract

The invention discloses a sea cucumber polysaccharide, natto and oligopeptide composition capable of efficiently lowering lipid, reducing plaques and inhibiting thrombus and application of the sea cucumber polysaccharide, natto and oligopeptide composition, and belongs to the field of application of active peptides. Comprising the following components: natto oligopeptide, pseudo-ginseng stem and leaf oligopeptide, cistanche oligopeptide (desert), fish white oligopeptide, L-arabinose, folium mori, yeast beta-glucan, epigallocatechin gallate (EGCG) and sea cucumber polysaccharide. The sea cucumber polysaccharide and the natto oligopeptide have a synergistic effect in a physiological environment; the composition has an excellent antithrombotic effect and excellent lipid-lowering and plaque-reducing effects, is beneficial to blood vessel health, and is simple in production process, low in cost and remarkable in economic benefit and social benefit.

Description

Technical Field

[0001] The present invention belongs to the field of active peptide preparation, and particularly relates to a sea cucumber polysaccharide natto oligopeptide composition with high efficiency in reducing lipid and plaque and inhibiting thrombosis, and its application. Background Art

[0002] Currently, commonly used thrombolytic drugs in clinical practice include urokinase, tissue-type plasminogen activator, streptokinase, etc. However, these drugs are expensive and have a short half-life. Moreover, these drugs can only be administered by injection, usually resulting in massive bleeding at the thrombus site and repeated thrombus formation. In 1986, Professor Hiroyuki Sumi investigated 173 kinds of foods and found that natto is the only food that can dissolve thrombus among them.

[0003] Natto contains various bioactive substances such as bioactive peptides, isoflavones, nattokinase, etc., and has functions such as dissolving thrombus, enhancing immunity, antioxidation, anti-cancer, antibacterial, preventing osteoporosis, weight loss, reducing blood lipid, relieving constipation, reducing blood pressure, etc. Through research, it has been found that the thrombolytic activity of nattokinase is 4 times that of plasmin, and the thrombolytic speed is 19 times that of urokinase. The high-efficiency thrombolytic effect of nattokinase is to reduce fibrinogen, promote the catalysis of plasminogen to convert into plasmin, and increase the synthesis of thrombus dissolution factors in the body. At the same time, due to the advantages of nattokinase such as oral administration, high safety, low price, and strong fibrinolytic activity, its development and research have become a research hotspot for thrombolytic products in recent years. Nattokinase is an alkaline protease. After oral administration, it must pass through the gastrointestinal tract, be absorbed by small intestinal epithelial cells and enter the blood, and then be transported to the whole body circulation before it can play its thrombolytic role.

[0004] Nattokinase is stable at pH 4.0 - pH 12.0, but will rapidly inactivate when the pH is less than 4.0. Pepsin in the stomach and the low pH environment will destroy the spatial structure of nattokinase, expose more pepsin binding sites and make it lose fibrinolytic activity, while the digestive environment in the intestine is milder than that in the stomach. If nattokinase is orally administered, it is necessary to ensure its activity and stability in the digestive system to achieve the purpose of thrombolysis.

[0005] The embedding technology is an effective method to stabilize nattokinase in the stomach and slowly release it in the intestine. Among the publicly disclosed Chinese invention patent documents, from the perspectives of preparation process, embedding method, and application efficacy, Chinese invention patent application CN117187331A discloses a preparation method of a nattokinase antioxidant active peptide and its beverage, a preparation method for obtaining specific active nattokinase peptides through enzymatic hydrolysis, but it does not involve the most prominent thrombus inhibition effect of nattokinase. At the same time, the acid-base environment during the enzymatic hydrolysis process and the high-temperature environment during enzyme inactivation are extremely likely to destroy the activity of nattokinase. Chinese invention patent application CN118325994A discloses nattokinase oligopeptides, their preparation methods, and their application in thrombolysis and blood pressure reduction. While retaining the thrombolytic activity of nattokinase in nattokinase, by releasing free L-arginine, the antithrombotic and blood pressure-reducing effects of the final product nattokinase oligopeptides are improved. However, it does not involve the method of maintaining activity that has received much attention. Chinese invention patent application CN 108719727 A discloses a solid beverage for conditioning cerebral thrombosis peptide compounding and its preparation method, using pleurotus eryngii peptide, yak bone peptide, antrodia camphorata peptide, seabuckthorn peptide, cordyceps militaris peptide, ginseng flower peptide, nattokinase peptide, chitin, walnut peptide, dictyophora indusiata peptide, oyster peptide, and cyclocarya paliurus polysaccharide as the main raw materials, with the efficacy of improving immune resistance and conditioning cerebral thrombosis, providing a direction for the development of scientifically nutritionally formulated peptide solid beverages. However, it does not involve the analysis of the effects of the main thrombus-inhibiting components.

[0006] Sea cucumber polysaccharides are important components of the body wall of sea cucumbers, mainly divided into two categories: one is fucosylated chondroitin sulfate of sea cucumbers, and the other is fucoidan of sea cucumbers. Although the constituent sugar groups of the two are different, some hydroxyl groups on the sugar chains are sulfated, and the content of sulfated polysaccharides is about 32% in both. The special structures of the two sea cucumber polysaccharides are unique to sea cucumbers. They have a variety of pharmacological activities, such as inhibiting thrombosis, anti-tumor, enhancing immunity, anticoagulation, inducing platelet aggregation, promoting fibrinolysis, anti-inflammatory, and lipid-lowering effects. Chinese Patent Application CN 109295135 A discloses an active compound oligopeptide solution and its preparation method and application. The content of oligopeptides with a molecular weight below 1000 Da is not less than 96%, and the crude polysaccharides contained have the effects of anti-blood coagulation, anti-thrombosis, enhancing the body's immunity, reducing blood sugar, and reducing blood lipids. However, the results only involve animal acute toxicity tests and do not involve thrombus inhibition pharmacodynamic tests. Chinese Patent CN 102028730 B discloses a compound sea cucumber product, its preparation method and its dosage form in use, which has anticoagulant and anti-thrombotic functions. However, the results only involve animal hemorheology tests and do not involve thrombus inhibition pharmacodynamic tests. Chinese Patent Application CN103830263A discloses the application of depolymerized sea cucumber glycosaminoglycan in pharmacy, and CN 103830264A discloses the application of depolymerized sea cucumber glycosaminoglycan in the preparation of drugs for preventing and treating thrombotic diseases, which can safely and effectively prevent and treat thrombotic diseases including deep vein thrombosis, thrombophilia, etc. However, the results only involve coagulation tests, and the thrombus inhibition rate is obtained through mechanical injury animal tests; there is no involvement in non-invasive animal tests that are more accurate for calculating the thrombus inhibition rate.

[0007] In addition, among the publicly disclosed Chinese invention patent documents, in terms of the efficacy analysis of compositions containing nattō ingredients, the Taiwanese patent with the invention patent publication number TWI441643B discloses a composition for regulating blood lipids and protecting the cardiovascular system, which includes rhodiola compound powder, red yeast rice, phytosterols, nattō, and vitamin B complex. It involves the blood lipid-lowering effects in animals and humans, but does not involve the most remarkable effects of nattō, such as inhibiting thrombosis, inhibiting the formation of thrombus, and promoting the reduction of thrombus plaques. Chinese invention patent application CN 117327756A discloses a nattō peptide, its preparation method, and applications. The prepared nattō peptide has good effects on freckle removal and blood lipid reduction. It involves the blood lipid-lowering effects in animals but does not involve the blood lipid-lowering and plaque-reducing effects of nattō peptide in humans. Chinese invention patent application CN 107029136A discloses a red yeast rice nattō product, its preparation method, and applications. Patients can reduce the lipid plaques in the arterial vessel wall by more than 20% in three months and by more than 40% in six months. The composition only involves red yeast rice and nattō and does not involve the synergistic sea cucumber polysaccharide. Chinese invention patent application CN106954798 A discloses red yeast rice nattō fine powder and its production method, which has good thrombolytic effects. It involves the thrombolytic effects in animals and the plaque-reducing effects in humans, but does not involve the lipid-lowering effects in animals and humans. Chinese invention patent application CN106798252 A discloses a nattō product with the effects of regulating the stomach, reducing blood lipids, and dissolving thrombus, which can comprehensively regulate blood lipids and protect the cardiovascular system. It involves the lipid-lowering and thrombolytic effects in animal experiments of nattō powder, red yeast rice, and oligosaccharides (fructose, isomaltose, galactose), but does not involve the lipid-lowering and plaque-reducing effects in humans. Chinese invention patent application CN 105343636 A discloses a medicated and edible composition with blood lipid-lowering functions and its preparation method. The composition contains nattō extract and chitosan oligosaccharide, which involves the health care effects of blood lipid reduction but does not involve the plaque-reducing effects. Chinese invention patent application CN 105030950A discloses a probiotic preparation for preventing and treating hyperlipidemia, its preparation method, and applications, which has preventive and adjuvant therapeutic effects on hyperlipidemia. It only involves the efficacy of preventing and treating hyperlipidemia and does not involve the plaque-reducing effects.

[0008] Chinese Patent Application CN 102823885 A discloses a sea cucumber chewable tablet and its preparation method. The sea cucumber chewable tablet has functions such as clearing the intestines and relieving constipation, reducing blood lipid, and reducing blood sugar. Chinese Patent Application CN 103445219 A discloses a nereis compound chewable tablet and its preparation method. The rich sea cucumber polysaccharide in it has a certain effect on improving human immunity, reducing blood sugar, and reducing blood lipid. Chinese Patent Application CN 103704713 A discloses a health food with sea cucumber's blood lipid-lowering function and its preparation method, which is effective for those with high blood lipid. Chinese Patent Application CN 107397226 A discloses a blood lipid-lowering sea cucumber polypeptide microcapsule and its preparation method, which has a specific blood lipid-lowering effect. Chinese Patent Application CN 108065014 A discloses a sea cucumber pressed candy, which has functions of clearing the intestines and relieving constipation, reducing blood lipid, and reducing blood sugar. Chinese Patent Application CN 115948492 A discloses a sea cucumber peptide with high sea cucumber polysaccharide content and its preparation method, which has the effects of enhancing self-immunity, antioxidant function, anti-fatigue, delaying aging, anticoagulation, anti-thrombosis, anti-tumor, and reducing blood lipid. Chinese Patent Application CN 117717168 A discloses a food composition that can reduce the three highs and its uses, which can achieve the purposes of reducing blood lipid, blood pressure, and blood sugar, tonifying qi and blood, promoting blood circulation and removing blood stasis, clearing human free radicals, and repairing damaged cells. Summary of the Invention

[0009] To solve the problem of improving the effect of a single component in reducing lipid plaques or inhibiting thrombosis, according to some embodiments of the present application, a sea cucumber polysaccharide natto oligopeptide composition, by weight, includes:

[0010] 1 - 6 parts of natto oligopeptide;

[0011] 0.5 - 2 parts of sea cucumber polysaccharide.

[0012] According to some embodiments of the present application, in a sea cucumber polysaccharide natto oligopeptide composition, 4 - 6 parts of natto oligopeptide and 1.5 - 2 parts of sea cucumber polysaccharide.

[0013] According to some embodiments of the present application, in a sea cucumber polysaccharide natto oligopeptide composition, 3 parts of natto oligopeptide and 0.5 part of sea cucumber polysaccharide.

[0014] According to some embodiments of the present application, in a sea cucumber polysaccharide natto oligopeptide composition, 5 parts of natto oligopeptide and 2 parts of sea cucumber polysaccharide.

[0015] According to some embodiments of the present application, a sea cucumber polysaccharide natto oligopeptide composition further includes:

[0016] 2 - 3 parts of panax notoginseng leaf oligopeptide;

[0017] 1 - 5 parts of cistanche oligopeptide (desert);

[0018] 3 - 4 parts of fish sperm oligopeptide.

[0019] According to a composition of sea cucumber polysaccharide nattokinase oligopeptide in some embodiments of the present application, among which, 2 parts of oligopeptide from Panax notoginseng stems and leaves, 4 parts of oligopeptide from Cistanche deserticola, and 3 parts of fish sperm oligopeptide.

[0020] According to a composition of sea cucumber polysaccharide nattokinase oligopeptide in some embodiments of the present application, among which, 3 parts of oligopeptide from Panax notoginseng stems and leaves, 3 parts of oligopeptide from Cistanche deserticola, and 4 parts of fish sperm oligopeptide.

[0021] According to a composition of sea cucumber polysaccharide nattokinase oligopeptide in some embodiments of the present application, further comprising

[0022] 2 - 3 parts of L - arabinose;

[0023] 1 - 2 parts of mulberry leaves;

[0024] 1 - 2 parts of yeast β - glucan;

[0025] 0.5 - 1 part of epigallocatechin gallate (EGCG).

[0026] According to a composition of sea cucumber polysaccharide nattokinase oligopeptide in some embodiments of the present application, among which, 2 parts of L - arabinose, 2 parts of mulberry leaves, 1 part of yeast β - glucan, and 0.5 part of epigallocatechin gallate (EGCG).

[0027] According to a composition of sea cucumber polysaccharide nattokinase oligopeptide in some embodiments of the present application, the particle size distribution of the nattokinase oligopeptide < 500 nm.

[0028] Use of the composition of sea cucumber polysaccharide nattokinase oligopeptide according to any one of some embodiments of the present application in the preparation of a drug for preventing or treating cardiovascular diseases or cerebrovascular diseases, or in the preparation of food or health products.

[0029] Use of the composition of sea cucumber polysaccharide nattokinase oligopeptide according to any one of some embodiments of the present application in the preparation of a drug for preventing or treating hyperlipidemia or vascular plaque or thrombus diseases, or in the preparation of food or health products.

[0030] (1) Oligopeptide from Panax notoginseng stems and leaves: contains various active ingredients and nutrients, such as saponins, flavonoids, vitamins, amino acids, etc.; has diverse pharmacological effects, including anti - anxiety, anti - depression, anti - osteoporosis, anti - diabetes, anti - cancer, liver protection, anti - aging, prevention of obesity, etc.

[0031] (2) Cistanche deserticola oligopeptide (desert): Cistanche deserticola contains rich nutritional components and bioactive components, such as phenyl ethanol glycosides, lignans, iridoids, sugars and their derivatives, etc., and has various effects such as regulating immunity, antioxidation, and anti-aging.

[0032] (3) Fish milt oligopeptide: The spermary of fish is also called fish milt, which is an organ for fish to produce and store sperm. Fish milt is an important pharmaceutical raw material. It contains a special protein called protamine, which has broad-spectrum antibacterial activity and important physiological functions such as hindering blood coagulation, assisting respiration, promoting digestion, lowering blood pressure, and inhibiting tumor growth.

[0033] (4) L-arabinose: L-arabinose is a non-caloric sugar. Although it has a similar taste to sucrose, its sweetness is only about half of that of sucrose. Its physiological function is mainly to selectively inhibit the enzymes related to sucrose metabolism in small intestine disaccharidase, thereby inhibiting the production efficiency of glucose and fructose, and thus reducing the amount of glucose and fructose absorbed by the small intestine. L-arabinose can control the accumulation of body fat caused by excessive sugar intake and the increase of triglycerides in the blood, and is of great significance in preventing obesity, regulating blood sugar and blood lipids, and controlling diabetes.

[0034] (5) Mulberry leaves: Mulberry leaves are rich in flavonoids, alkaloids, amino acids, organic acids and vitamins, etc., and have effects such as lowering blood sugar, lowering blood pressure, lowering blood lipids, and delaying aging.

[0035] (6) Yeast β-glucan: Yeast β-glucan is a natural polysaccharide from yeast cell wall, and has various biological activities such as enhancing immunity, anti-tumor, anti-infection, anti-inflammatory, improving intestinal health, lowering blood sugar, lowering cholesterol, and promoting wound healing. It has been used in industries such as medicine, cosmetics and food processing.

[0036] (7) Epigallocatechin gallate (EGCG): Epigallocatechin gallate is the catechin with the highest content in green tea, and has activities such as antibacterial, antiviral, antioxidation, anti-aging, anti-inflammatory and anti-cancer. It is applied in fields such as food health care, antibacterial, fresh-keeping and cosmetics.

[0037] Beneficial effects:

[0038] In the first aspect, the experimental results show that the combination of sea cucumber polysaccharide and nattokinase oligopeptide has a synergistic effect on anti-thrombosis in a physiological environment, and the synergistic effect achieves an efficient effect of inhibiting thrombosis. The synergistic effect of sea cucumber polysaccharide and nattokinase oligopeptide is due to the following: Sea cucumber polysaccharide has the effect of anti-thrombin and, more importantly, the effect of promoting fibrinolysis. Therefore, sea cucumber polysaccharide can increase the activity of plasmin, directly degrade fibrinogen, significantly reduce fibrin in the fibrin gel and make it easily removed by plasmin, thereby inhibiting the aggregation function of fibrinogen, affecting the monomer aggregation process, changing the fibrin gel structure, and thus enhancing its sensitivity to plasmin and making it easily removed. At the same time, nattokinase oligopeptide also has the plasmin activity of nattokinase, which can reduce fibrinogen, promote the conversion of plasminogen to plasmin, and especially increase the synthesis of thrombolytic factors in the body. The anticoagulation of sea cucumber polysaccharide has the effect of preventing thrombosis, and its effect of promoting fibrinolysis combined with the effect of nattokinase oligopeptide in promoting fibrinolysis and increasing the synthesis of thrombolytic factors in the body can effectively clear thrombolysis, acting together from two dimensions of preventing thrombosis formation to thrombolysis, achieving the synergistic effect of anti-thrombosis.

[0039] In the second aspect, the experimental results show that the combination of sea cucumber polysaccharide and nattokinase oligopeptide has a synergistic effect on lipid-lowering and plaque reduction in a physiological environment, and the synergistic effect achieves an efficient effect of lipid-lowering and plaque reduction. The synergistic effect of sea cucumber polysaccharide and nattokinase oligopeptide in lipid-lowering and plaque reduction is due to the following: Sea cucumber polysaccharide contains rich sulfate groups in its structure and has the effects of inhibiting thrombosis formation, enhancing immune function, regulating vascular smooth muscle cells and endothelial cells, and reducing blood viscosity. At the same time, sea cucumber polysaccharide can increase the activity of plasmin to promote fibrinolysis, increase the activity of plasmin, directly degrade fibrinogen, significantly reduce fibrin in the fibrin gel and make it easily removed by plasmin, and inhibit the aggregation function of fibrinogen, affecting the monomer aggregation process, changing the fibrin gel structure, and thus enhancing its sensitivity to plasmin and making it easily removed, enhancing the effect of dissolving plaques. Nattokinase oligopeptide has the plasmin activity of nattokinase, and the highly efficient thrombolytic effect of nattokinase can reduce fibrinogen, promote the conversion of plasminogen to plasmin, increase the synthesis of thrombolytic factors in the body, promote the dissolution of thrombus in blood vessels, inhibit the formation of thrombus, and promote the reduction of thrombus plaques. Description of the Drawings

[0040] Figure 1 Thrombosis formation rate in mouse tails;

[0041] Figure 2 Relative length of thrombosis formation in mice;

[0042] Figure 3Mouse tail thrombus sections (A. normal group; B. thrombus group; C. nattokinase group; D. natto oligopeptide group; E. sea cucumber polysaccharide group; F. sea cucumber polysaccharide - natto oligopeptide group). Detailed implementation manners

[0043] The present invention will be further described below through examples. It should be noted that the examples do not constitute a limitation on the scope of protection required by the present invention.

[0044] A. Term description:

[0045] Natto peptide: Natto peptide is an active ingredient extracted from natto. Natto is a fermented soybean product made by fermenting soybeans with Bacillus subtilis. Natto peptide is rich in various nutrients, including nattokinase, natto bacteria, high - quality small - molecule proteins (peptides), superoxide dismutase, biological polysaccharides, isoflavones, saponins, lecithin, vitamin E, pyridine dicarboxylic acid, vitamin K2 and other hundreds of physiologically active substances beneficial to the human body.

[0046] Natto oligopeptide: Natto oligopeptide is a mixed peptide product composed of 2 - 6 amino acids with a molecular weight of 200 - 800 Dalton.

[0047] Natto oligopeptide can be prepared in the following way:

[0048] S1. Take 100 g of natto freeze - dried powder prepared by fermenting soybeans with natto bacteria, dissolve it in 1000 mL of normal saline. The natto freeze - dried powder solution passes through a 29000 - molecular - weight ultrafiltration membrane, and the permeate is taken to obtain components with a molecular weight lower than 29000. The permeate passes through a 26000 - molecular - weight ultrafiltration membrane, and the retentate is taken to obtain components with a molecular weight higher than 26000 but lower than 29000 as nattokinase component F1. The first ultrafiltration retentate and the second ultrafiltration permeate are combined as natto component F2.

[0049] S2. The solution of natto component F2 is hydrolyzed with digestive enzymes such as trypsin for 4 hours to obtain a natto hydrolysate (hydrolysis pH is 8.5, hydrolysis temperature is 50 °C). The purpose of hydrolysis is to decompose proteins into small peptides (small molecular peptides, usually composed of 2 to 20 amino acid residues) by digestive enzymes. The hydrolysate is analyzed for peptide segments by LC-MS / MS and De Novo de novo sequencing method. The analysis conditions are as follows: Chromatographic column: C18, 3μm, 75μm×15cm; Mobile phase: A, 0.1% Formic acid in water, B, 0.1% Formic acid in 80% Acetonitrile / H2O; Chromatographic gradient: starting from 10% strong solvent concentration, linearly increasing to 25%, 50%, 75% and 90%; Spray voltage: 2.0 kV; Capillary temperature: 320 °C; RF Lens: 40; Resolution setting: primary 120,000@m / z 200, secondary 30,000@m / z 200; Parent ion scan range: m / z 350 - 1550; Daughter ion scan range: starting from m / z110, fragmentation mode: HCD.

[0050] The raw data collected by mass spectrometry is analyzed for de novo sequencing using the De Novo data analysis software PEAKS. The software parameters are set as follows: Enzyme: No-specific, Variable modification: Oxidation(M), Deamidated(N, Q), Peptide mass tolerance: ±10 ppm, Fragment mass tolerance: 0.02 Da. (Enzyme: non-specific, variable modification: oxidation (M), deamidation (N, Q), peptide mass tolerance: ±10 ppm, fragment mass tolerance: 0.02 Da). The types and quantities of amino acid residues in front of glycine and the types and quantities of amino acid residues in front of arginine are respectively counted. It can be seen that the peptide bonds of tyrosine-glycine, phenylalanine-glycine, tyrosine-arginine, the peptide bond at the carboxyl terminus of lysine, and the peptide bond at the carboxyl terminus of arginine are used as the target cleavage sites to hydrolyze natto component F2. The hydrolysis conditions are as follows: Trypsin: Chymotrypsin = 1:1 - 3, enzyme addition amount: 1 - 3%, hydrolysis temperature: 37 - 45 °C, hydrolysis pH: 7.5 - 8.5, hydrolysis time 3 - 6 hours, to obtain a second hydrolysate.

[0051] S3. The second hydrolysate is separated by a nanofiltration membrane with a cut-off of 2000 Da, and the permeate is collected to obtain the natto hydrolysate component F3, that is, oligopeptides with a molecular weight lower than 2000 Da are obtained.

[0052] S4. Use an SP Sepharose High Performance strong cation exchange column and equilibrate the cation exchange column with 20 mmol / L PB (pH 6.0). After sample loading and re-equilibration of the present invention, perform elution with a 0 - 40 min: 0 - 1 M NaCl concentration gradient, and collect the eluate with a retention time of 11 - 19 min as the nattokinase hydrolysate component F4.

[0053] S5. Thoroughly mix the nattokinase hydrolysate component F4 and the nattokinase component F1, and then perform freeze-drying to obtain nattokinase oligopeptide powder.

[0054] Specifically, it can be prepared by the method of Example 1 in CN 118325994 B, Nattokinase Oligopeptide, Its Preparation Method and Application in Thrombolysis and Blood Pressure Reduction.

[0055] Sea cucumber polysaccharide: Sea cucumber polysaccharide is an important component in the body wall of sea cucumbers, and its content can account for more than 6% of the total organic matter of dry sea cucumbers. There are mainly two types of polysaccharides in the body wall of sea cucumbers: one is holothurian glycosaminoglycan (HG), and the other is holothurian fucan (HF). HG is a branched heteropolysaccharide composed of D-N-acetylgalactosamine, D-glucuronic acid, and L-fucose, while HF is a linear homogeneous polysaccharide composed of L-fucose. Some hydroxyl groups on both sugar chains are sulfated, and the sulfate ester groups account for about 32% of the polysaccharide content. Currently, sea cucumber polysaccharide is mainly obtained by the separation and purification of sea cucumber raw materials, and the preparation technology is as follows: raw material pretreatment → enzymatic hydrolysis → centrifugal separation → ultrafiltration and concentration → freeze-drying → passing the crude sea cucumber polysaccharide powder through a column → drying to obtain refined sea cucumber polysaccharide.

[0056] Sea cucumber polysaccharide can be prepared in the following manner:

[0057] S1. Raw material pretreatment: Take sea cucumber raw materials to make pulp to obtain sea cucumber pulp, and measure the total protein content in the sea cucumber pulp using the Kjeldahl method;

[0058] S2. Enzymatic hydrolysis: Add 5 - 8 times the volume of water to the sea cucumber pulp obtained in step S1, mix evenly, add a 10% NaOH solution by mass concentration to adjust the pH to 6.5 - 8.0, add a compound protease accounting for 1 - 3% of the total protein mass in the sea cucumber pulp, and perform enzymatic hydrolysis treatment at 45 - 60 °C for 2 - 6 hours to obtain a sea cucumber enzymatic hydrolysate; the compound protease is a compound of trypsin, pepsin, flavor protease, and collagenase, and the mass ratio of trypsin, pepsin, flavor protease, and collagenase = 1 - 3: 1 - 3: 1 - 2: 2 - 7;

[0059] S3. Centrifuge the sea cucumber enzymolysis solution obtained in step S2 at 10,000 - 16,000 revolutions per minute using a tubular centrifuge, and collect the centrifuged supernatant;

[0060] S4. Repeatedly concentrate and filter the supernatant collected in step S3 using an ultrafiltration membrane with a molecular weight cut-off of 3,000 - 8,000 Da, and collect the final concentrated solution, which is the concentrated sea cucumber polysaccharide solution; after each concentration and filtration, add water with a volume half of the remaining concentrated solution and continue concentration and filtration, and repeat the concentration and filtration 3 - 5 times;

[0061] S5. Freeze-dry the concentrated sea cucumber polysaccharide solution obtained in step S4 to obtain crude sea cucumber polysaccharide powder;

[0062] S6. Pack the crude sea cucumber polysaccharide powder prepared in step S5 into a circular fiberglass resin column. First, pass through the column with alcohol having a volume 2 - 5 times the volume of the column filled with the crude sea cucumber polysaccharide powder and a volume concentration of 65%, and then pass through the column with alcohol having a volume 1 - 3 times the volume of the column filled with the crude sea cucumber polysaccharide powder and a volume concentration of 95%; the diameter-to-height ratio of the resin column is 1:10 - 15, and the filling amount of the crude sea cucumber polysaccharide powder is 65 - 85% of the volume of the resin column;

[0063] S7. Take out the sea cucumber polysaccharide in the resin column, place it in a silica gel drying oven to dry and remove alcohol, and obtain refined sea cucumber polysaccharide.

[0064] Specifically, it can be prepared by the method of Example 3 in CN 106117382 B, a method for separating and refining sea cucumber polysaccharide.

[0065] The nattokinase oligopeptide and sea cucumber polysaccharide prepared by other methods are also applicable to the present invention.

[0066] Nattokinase: Nattokinase (NK) is a serine protease produced by Bacillus subtilis natto during the natto fermentation process. Nattokinase is an enzyme with strong fibrinolytic activity, which can significantly dissolve thrombi in vivo and in vitro, significantly shorten the euglobulin lysis time, and can stimulate venous endothelial cells to produce plasminogen activator (t-PA), thereby more effectively exerting the thrombolytic effect.

[0067] B. Description of experimental materials:

[0068] The sea cucumber polysaccharide is produced by Dalian Deep Blue Peptide Technology R & D Co., Ltd. (Dalian, China).

[0069] The nattokinase oligopeptide is produced by Dalian Deep Blue Peptide Technology R & D Co., Ltd. (Dalian, China).

[0070] Nattokinase (20,000 FU, RZ-NDJM) is purchased from Xi'an Rongzhen Biotechnology Co., Ltd. (Xi'an, China).

[0071] Carrageenan (X110772A) was purchased from Shanghai Xianding Biotechnology Co., Ltd. (Shanghai, China).

[0072] C. Examples:

[0073] Example C1: A sea cucumber polysaccharide natto oligopeptide composition C1 for the prevention and treatment of thrombosis, which is made of the following components by weight: compound peptide powder (3 parts of natto oligopeptide, 2 parts of Panax notoginseng stem and leaf oligopeptide, 4 parts of desert cistanche oligopeptide, 3 parts of fish sperm oligopeptide), 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, 0.5 part of epigallocatechin gallate (EGCG), and 0.5 part of sea cucumber polysaccharide.

[0074] Example C2: A sea cucumber polysaccharide natto oligopeptide composition C1 for the prevention and treatment of hyperlipidemia, which can reduce blood lipid and plaque formation. It is made of the following components by weight: compound peptide powder (5 parts of natto oligopeptide, 3 parts of Panax notoginseng stem and leaf oligopeptide, 3 parts of desert cistanche oligopeptide, 4 parts of fish sperm oligopeptide), 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, 0.5 part of epigallocatechin gallate (EGCG), and 0.5 part of sea cucumber polysaccharide.

[0075] D. Comparative Examples:

[0076] Comparative Example D1: A sea cucumber polysaccharide composition D1, which is made of the following components by weight: compound peptide powder (2 parts of Panax notoginseng stem and leaf oligopeptide, 4 parts of desert cistanche oligopeptide, 3 parts of fish sperm oligopeptide), 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, 0.5 part of epigallocatechin gallate (EGCG), and 0.5 part of sea cucumber polysaccharide.

[0077] Comparative Example D2: A natto oligopeptide composition D2, which is made of the following components by weight: compound peptide powder (3 parts of natto oligopeptide, 2 parts of Panax notoginseng stem and leaf oligopeptide, 4 parts of desert cistanche oligopeptide, 3 parts of fish sperm oligopeptide), 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, and 0.5 part of epigallocatechin gallate (EGCG).

[0078] Comparative Example D3: A sea cucumber polysaccharide composition D3, which is made of the following components by weight: compound peptide powder (3 parts of Panax notoginseng stem and leaf oligopeptide, 3 parts of desert cistanche oligopeptide, 4 parts of fish sperm oligopeptide), 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, 0.5 part of epigallocatechin gallate (EGCG), and 0.5 part of sea cucumber polysaccharide.

[0079] Comparative Example D4: A sea cucumber polysaccharide composition D4, which is made of the following components by weight: compound peptide powder (5 parts of natto oligopeptide, 3 parts of Panax notoginseng stem and leaf oligopeptide, 3 parts of cistanche oligopeptide (desert), 4 parts of fish sperm oligopeptide), 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, 0.5 part of epigallocatechin gallate (EGCG).

[0080] Table 1 Comparison of Components of Example C1 and Comparative Examples D1 - D2

[0081]

[0082]

[0083] Table 2 Comparison of Components of Example C2 and Comparative Examples D3 - D4

[0084]

[0085] Note: "×" in the above table indicates not added.

[0086] E. Experimental Instructions:

[0087] E1. Animal Experiments on Sea Cucumber Polysaccharide Natto Oligopeptide Composition C1, Sea Cucumber Polysaccharide Composition D1, and Natto Oligopeptide Composition D2

[0088] Animal Grouping and Administration: 48 KM mice were randomly divided into 6 groups (see Table 3) after 7 days of adaptive feeding. According to the grouping, each mouse was intragastrically administered the corresponding dose of the reagent for 8 consecutive days. The body weight was recorded daily, and the changes in the appearance, hair, behavioral activities, and mental state of the animals were observed.

[0089] Table 3 Animal Grouping and Administration Information

[0090]

[0091] Animal Modeling: After 6 days of administration, a 2.5% (w / v) carrageenan solution was prepared and intraperitoneally injected at a dose of 0.1 mL / 10 g. At the same time, the bedding was replaced with corncob bedding, and the temperature of the animal room was adjusted to 18°C. The tail thrombus model of the mice was successfully established after 48 hours.

[0092] Thrombus Rate Measurement: On the 8th day, 2 hours after the last administration, the mice were fixed, and the black tail condition of the mice was photographed with a mobile phone to observe the thrombus formation rate in the tails of the mice (see Figure 1 ). At the same time, a ruler was used to measure the total length and black tail length of the mouse tail to calculate the relative length of thrombus formation (see Figure 2 ).

[0093] Thrombus Formation Rate (%) = Number of Mice with Thrombus / Total Number of Mice × 100%

[0094] Relative length of thrombosis (%) = total length of thrombosis in mice / length of mouse tail × 100%

[0095] After one week of adaptive feeding of mice, normal saline, nattokinase, and each formula group were respectively administered to each group of mice by gavage according to the dosing regimen. Each group was administered for 2 days every day for 8 consecutive days. 2 hours after the last administration on the 6th day, the total tail length of the mice was measured, and 0.1 mL / kg of 1% carrageenan was injected intraperitoneally to establish a model. 48 hours after modeling, paraffin sections were made at 7.5 cm from the tail of the mice, and the tails of mice in different treatment groups were stained with H&E (see Figure 3 ).

[0096] E2. Animal experiments on the composition C2 of sea cucumber polysaccharide nattokinase oligopeptide, the composition D3 of sea cucumber polysaccharide, and the composition D4 of nattokinase oligopeptide:

[0097] Animal grouping and dosing: 48 KM mice were randomly divided into 6 groups (see Table 4), namely the normal group, the model control group, and the dose groups (nattokinase group, nattokinase oligopeptide group, sea cucumber polysaccharide group, sea cucumber polysaccharide nattokinase oligopeptide group). Using C57BL / 6 pure-line ApoE - / - mice as materials, they were fed with a high-cholesterol diet for 12 weeks, and the atherosclerotic plaque mouse model was successfully established. Observe and record the appearance, hair, behavior, and mental state changes of the animals, etc.

[0098] Table 4 Animal grouping and dosing information

[0099]

[0100] E2.1. Observation of carotid artery pathological morphology (H&E staining and Masson staining): For ApoE - / - mice with successful high-fat diet-induced modeling, the carotid artery vessels were cut and paraffin-embedded and sectioned, then H&E staining was completed, and morphological observation was carried out under a microscope after sealing the slides. The Image Pro Plus image processing software was used to measure the area of carotid artery plaque formation. In addition, Masson collagen fiber staining was performed to observe the plaque morphology, and the thickness of the plaque fibrous cap was quantitatively analyzed (the fibrous cap is defined as the non-necrotic core area on the lumen side of the plaque, and three random sites were selected for measurement in each specimen, and the average value was taken). The analysis results of the fibrous cap thickness are shown in Table 5.

[0101] Table 5 Analysis of fibrous cap thickness

[0102] Serial number Group Fiber cap thickness (μm) 1 Normal group 56.1±2.23 2 Model control group 64.6±4.07 3 Nattokinase group C3 52.2±3.17 4 Natto oligopeptide composition D4 51.4±2.80 5 Sea cucumber polysaccharide composition D3 50.7±2.15 6 Sea cucumber polysaccharide natto oligopeptide composition C2 47.0±3.63

[0103] Note: There were statistically significant differences in the comparison of fibrous cap thickness among groups, P < 0.05.

[0104] E2.2. Fat staining in tissue (oil red staining): Weigh 0.5g of oil red dry powder and dissolve it in 100ml of isopropanol to prepare oil red storage solution, take out the carotid artery OCT block in the -80℃ refrigerator, prepare 6μm slices, and put them in a 55℃ oven for 1h. Prepare oil red working solution according to the ratio of 3:2 of oil red storage solution: double distilled water, filter it 3 times with filter paper and let it stand for 10min for staining. Then put the slices in PBS buffer to wash away OCT, put the slices in 60% isopropanol for 10min, and then put the slices in oil red working solution to avoid light, and stain at 37℃ for 30min; put them in 60% isopropanol until the interstitium is clear; put them in hematoxylin staining solution to stain the nucleus for 10s, and wash them continuously with water until the excess stain is washed away. The results of lipid content analysis are shown in Table 6.

[0105] Table 6 Comparison of the percentage of lipid in the carotid artery plaque area in mice

[0106] Serial number Group Percentage of lipid in plaque area (%) 1 Normal group 3.30±0.37 2 Model control group 2.21±0.30 3 Nattokinase group C3 3.71±0.39 4 Natto oligopeptide composition D4 3.95±0.41 5 Sea cucumber polysaccharide composition D3 4.12±0.33 6 Sea cucumber polysaccharide natto oligopeptide composition C2 4.76±0.56

[0107] Note: There were significant differences in the percentage of carotid artery plaque area occupied by lipids among the groups, P < 0.05.

[0108] E2.3. Blood lipid determination: About 3 mL of mouse medial canthal artery blood was collected and centrifuged at 3000 r / min to obtain the supernatant. The levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) were determined using an automatic biochemical analyzer. The results of mouse blood lipid detection are shown in Table 7.

[0109] Table 7 Measurement results of blood lipid levels in the common carotid artery of mice (mmol / L, )

[0110]

[0111] E3. Clinical trials of sea cucumber polysaccharide and natto oligopeptide composition C2, sea cucumber polysaccharide composition D3, and natto oligopeptide composition D4:

[0112] E3.1. Methods and procedures for product effect verification: Natto oligopeptides, sea cucumber polysaccharides, and sea cucumber polysaccharide natto oligopeptides were distributed to three groups of volunteers according to the inclusion and exclusion criteria. Transcranial Doppler ultrasound and left and right carotid artery color Doppler imaging examinations were performed at baseline and 30 days after oral administration of the test product to evaluate changes in carotid artery plaque area and media thickness; and venous blood was collected for blood lipid examination. The methods and procedures are shown in Table 8.

[0113] Table 8 Methods and procedures for verifying the effects of taking sea cucumber polysaccharide natto oligopeptide products

[0114]

[0115] Inspection Criteria for Verifying the Efficacy of Product Administration: After taking the product containing sea cucumber polysaccharide nattokinase oligopeptide, inspections of carotid artery plaques, intima-media thickness, and blood lipids are carried out. The evaluation criteria for the above three inspections are shown in Table 9.

[0116] Table 9 Evaluation Criteria for Inspections

[0117]

[0118] 69 volunteers from 14 regions across the country were divided into 3 groups and took nattokinase oligopeptide, sea cucumber polysaccharide, and the product containing sea cucumber polysaccharide nattokinase oligopeptide respectively. After 30 days, the improvement of blood lipids and carotid artery plaques / intima-media thickness was statistically compared with the baseline. The effective rates of the above three products after administration are shown in Table 9', Table 10, and Table 11.

[0119] Table 9' Effective Rate of Lipid Lowering and Plaque Reduction after Taking Nattokinase Oligopeptide Composition D4

[0120]

[0121] Table 10 Effective Rate of Lipid Lowering and Plaque Reduction after Taking Sea Cucumber Polysaccharide Composition D3

[0122]

[0123] Table 11 Effective Rate of Lipid Lowering and Plaque Reduction after Taking Sea Cucumber Polysaccharide Nattokinase Oligopeptide Composition C2

[0124]

[0125] F. Analysis of Experimental Results:

[0126] According to Figure 1 - 2 , Figure 1 it shows that no thrombus forms at the tail of mice in the normal group, and the thrombus rate of the other 5 groups of mice is 100%. According to Figure 2 , after statistics, the thrombus inhibition rate of mice in the sea cucumber polysaccharide nattokinase oligopeptide group is 1.1 times that of the sea cucumber polysaccharide group, 1.5 times that of the nattokinase oligopeptide group, and 2.3 times that of the nattokinase group. Figure 2 The results of

[0127] According to Figure 3, the caudal artery of the normal group was flat and there was no thrombus in the lumen; however, after the carrageenan-induced thrombosis model was established, it was found that the caudal artery vessels at 7.5 cm were almost completely occupied by thrombus. After treatment with nattokinase, natto oligopeptide composition D2, sea cucumber polysaccharide composition D1, and sea cucumber polysaccharide natto oligopeptide composition C1, it was found that the proportion of thrombus in the vascular lumen decreased to varying degrees. Among them, the thrombus in the sea cucumber polysaccharide natto oligopeptide group almost completely disappeared, which also proved that it had the best thrombus inhibitory effect.

[0128] As can be seen from the results in Table 5, the fibrous cap thickness was model control group > normal group > dose groups (nattokinase group, natto oligopeptide group, sea cucumber polysaccharide group, sea cucumber polysaccharide natto oligopeptide group). And the histological morphological characteristics of unstable carotid plaques were less collagen fiber content, higher lipid content, and thinner fibrous cap. The stability of carotid plaques in mice was in the order of model control group > normal group > dose groups (nattokinase group, natto oligopeptide group, sea cucumber polysaccharide group, sea cucumber polysaccharide natto oligopeptide group), indicating that the dose groups (nattokinase group, natto oligopeptide group, sea cucumber polysaccharide group, sea cucumber polysaccharide natto oligopeptide group) could cause carotid plaque destabilization. The effects were in the order of sea cucumber polysaccharide natto oligopeptide group > sea cucumber polysaccharide group > natto oligopeptide group > nattokinase group.

[0129] As can be seen from the results in Table 6, the percentage of lipid in the plaque area was dose groups > control group > model control group, and the histological morphological characteristics of unstable carotid plaques were higher lipid content and thinner fibrous cap. It shows that the dose groups (nattokinase group, natto oligopeptide group, sea cucumber polysaccharide group, sea cucumber polysaccharide natto oligopeptide group) can cause carotid plaque destabilization. The effects were in the order of sea cucumber polysaccharide natto oligopeptide group > sea cucumber polysaccharide group > natto oligopeptide group > nattokinase group.

[0130] As can be seen from the results in Table 7, compared with the nattokinase group, the sea cucumber polysaccharide natto oligopeptide group had excellent effects in reducing blood lipids (triglyceride, total cholesterol, low-density lipoprotein).

[0131] The results in Tables 9'-11 showed that natto oligopeptide had fibrinolytic activity; sea cucumber polysaccharide significantly enhanced the fibrinolytic activity, and its synergistic effect with natto oligopeptide could achieve the effects of highly reducing blood lipids and reducing plaques.

[0132] In the present invention, the focus of various active ingredients is different. By combining various components together, especially sea cucumber polysaccharide and natto oligopeptide (with nattokinase and plasmin activities) having a synergistic effect in the physiological environment, the formulated special dietary food has excellent anti-thrombotic effects and is beneficial to vascular health; at the same time, the production process is simple, the cost is low, and the economic and social benefits are remarkable.

[0133] The saponin, lecithin, isoflavone and α-tocopherol in the main component natto of the present invention have the effect of lowering blood lipids (lowering triglycerides, total cholesterol and low-density lipoprotein); nattokinase has fibrinolytic protease activity, which can promote thrombus dissolution in blood vessels, inhibit the formation of thrombus, and promote the reduction of thrombus plaques; nattokinase can promote metabolism, reduce blood lipid levels, and reduce plaque formation. Sea cucumber polysaccharide contains rich sulfate groups in its structure, which has the effects of inhibiting thrombosis, enhancing immune function, regulating vascular smooth muscle cells and endothelial cells, and reducing blood viscosity. At the same time, sea cucumber polysaccharide can increase fibrinolytic enzyme activity to promote fibrinolysis and enhance the effect of dissolving plaques. The preparation process of the composition of the present invention is simple, the bioavailability is high, the sea cucumber polysaccharide and natto oligopeptide synergize, have excellent lipid-lowering and plaque-reducing effects, are beneficial to vascular health, and have broad application prospects.

[0134] 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 sea cucumber polysaccharide natto oligopeptide composition, characterized in that: By weight, including: 1-6 portions of natto oligopeptides; 0.5-2 parts of sea cucumber polysaccharide.

2. The sea cucumber polysaccharide natto oligopeptide composition according to claim 1, characterized in that: in, 4-6 parts of natto oligopeptides and 1.5-2 parts of sea cucumber polysaccharides.

3. The sea cucumber polysaccharide natto oligopeptide composition according to claim 1, characterized in that: in, 3 parts of natto oligopeptides and 0.5 parts of sea cucumber polysaccharides.

4. The sea cucumber polysaccharide natto oligopeptide composition according to claim 1, characterized in that: in, 5 parts of natto oligopeptides and 2 parts of sea cucumber polysaccharides.

5. The sea cucumber polysaccharide natto oligopeptide composition according to any one of claims 1 to 4, characterized in that: Also includes: 2-3 parts of Panax notoginseng stem and leaf oligopeptide; Cistanche deserticola oligopeptide (desert) 1-5 parts; 3-4 parts of milt oligopeptide.

6. The sea cucumber polysaccharide natto oligopeptide composition according to claim 5, characterized in that: in, 2 parts of Panax notoginseng stem and leaf oligopeptide, 4 parts of Cistanche deserticola oligopeptide (desert), and 3 parts of milt oligopeptide.

7. The sea cucumber polysaccharide natto oligopeptide composition according to claim 5, characterized in that: in, 3 parts of Panax notoginseng stem and leaf oligopeptide, 3 parts of Cistanche deserticola oligopeptide (desert), and 4 parts of milt oligopeptide.

8. The sea cucumber polysaccharide natto oligopeptide composition according to claim 1 or 5, characterized in that: Also includes 2-3 parts of L-arabinose; 1-2 portions of mulberry leaves; Yeast β-glucan 1-2 parts; Epigallocatechin gallate (EGCG) 0.5-1 part.

9. The sea cucumber polysaccharide and natto oligopeptide composition according to claim 6, characterized in that: in, 2 parts of L-arabinose, 2 parts of mulberry leaves, 1 part of yeast β-glucan, and 0.5 parts of epigallocatechin gallate (EGCG).

10. The sea cucumber polysaccharide and natto oligopeptide composition according to claim 1, characterized in that: The particle size distribution of the natto oligopeptide is less than 500 nm.

11. Use of the sea cucumber polysaccharide and natto oligopeptide composition according to any one of claims 1 to 10 in the preparation of a drug for preventing or treating cardiovascular disease or cerebrovascular disease, or in the preparation of food or health products.

12. Use of the sea cucumber polysaccharide and natto oligopeptide composition according to any one of claims 1 to 8 in the preparation of a drug for preventing or treating hyperlipidemia or vascular plaque or thrombotic diseases, or in the preparation of food or health products.

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