Pumpkin seed protein peptide for improving vascular endothelial cell dysfunction and preparation method thereof

Abstract

The invention provides a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction and a preparation method thereof, and relates to the technical field of bioactive peptides, the pumpkin seed protein peptide is obtained by enzymolysis of pumpkin seed crude protein with compound protease; the compound protease comprises alkaline protease and trypsin; the mass ratio of the alkaline protease to the trypsin is (1: 3)-(3: 1), and compared with other enzymolysis modes, the pumpkin seed protein peptide prepared by performing enzymolysis on the pumpkin seed crude protein by adopting the alkaline protease and the trypsin has the advantage that the capability of scavenging peroxide free radicals is improved; and the arginine is enriched. The form of peptide in the pumpkin seed protein peptide conforms to the characteristics of high-activity antihypertensive active peptide; the traditional Chinese medicine composition has a relatively good improvement effect on vascular endothelial cell dysfunction and can promote the generation of NO factors. The technical problem that in the prior art, no bioactive peptide capable of improving vascular endothelial cell dysfunction exists is solved.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, in particular to a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction and a preparation method thereof. Background Art

[0002] Vascular endothelial cells are the mechanical barrier between vascular smooth muscle and blood, and are the target organs of many active substances. They can secrete vasodilator factors and vasoconstrictor factors. The balance between the two can regulate vasoconstriction, promote the balance between the fibrinolytic system and coagulation, inhibit platelet aggregation and inflammatory cell adhesion to the vascular endothelium, and regulate the growth of vascular smooth muscle. Vascular endothelial dysfunction is specifically manifested as reduced NO production or decreased utilization, vascular permeability disorders and increased ROS production, which ultimately lead to the occurrence of adverse consequences such as impaired vasoconstriction, proinflammatory state, thrombosis, oxidative stress and arterial wall proliferation.

[0003] Studies have shown that endothelial cell dysfunction is a key factor in the occurrence of diseases such as hypertension, diabetes, atherosclerosis and heart failure, and improving endothelial dysfunction plays an important role in the prevention and treatment of cardiovascular diseases. However, there are currently very few drugs for improving endothelial dysfunction.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction, so as to solve the technical problem of the lack of bioactive peptides that can be used to improve vascular endothelial cell dysfunction in the prior art.

[0006] The second object of the present invention is to provide a method for preparing pumpkin seed protein peptide for improving vascular endothelial cell dysfunction.

[0007] The third object of the present invention is to provide the use of the pumpkin seed protein peptide or the pumpkin seed protein peptide prepared by the above preparation method in the preparation of drugs for preventing or treating vascular endothelial cell dysfunction.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction, wherein the pumpkin seed protein peptide is obtained by enzymatic hydrolysis of pumpkin seed crude protein by a composite protease;

[0010] The composite protease comprises alkaline protease and trypsin;

[0011] The mass ratio of the alkaline protease to trypsin is 1:3 to 3:1.

[0012] Furthermore, the mass ratio of alkaline protease to trypsin is 1:2 to 2:1.

[0013] Furthermore, the added amount of the composite protease is 0.2-1.1% of the total mass of pumpkin seed crude protein.

[0014] In a second aspect, the present invention provides a method for preparing a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction, comprising the following steps:

[0015] A. Adding compound protease to pumpkin seed crude protein homogenate for enzymolysis;

[0016] B. The enzymatic hydrolysate prepared in step A is heated to inactivate the enzyme, the supernatant is obtained by centrifugation, and the supernatant is concentrated and dried to obtain pumpkin seed protein peptide.

[0017] Furthermore, the method for preparing the pumpkin seed crude protein homogenate comprises adding water to the pumpkin seed crude protein and homogenizing to obtain the pumpkin seed crude protein homogenate.

[0018] Furthermore, the mass ratio of the pumpkin seed crude protein to water is 1:7.33-15.67.

[0019] Furthermore, the enzymatic hydrolysis pH of the pumpkin seed crude protein homogenate is 7.5-10.

[0020] Furthermore, the temperature of the enzymatic hydrolysis is 48-57°C.

[0021] Furthermore, the enzymatic hydrolysis time is 3 to 6 hours.

[0022] In a third aspect, the present invention provides the use of the above-mentioned pumpkin seed protein peptide or the pumpkin seed protein peptide prepared by the above-mentioned preparation method in the preparation of a product for preventing or treating vascular endothelial cell dysfunction.

[0023] The present invention provides a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction. The pumpkin seed protein peptide is prepared by enzymatic hydrolysis of pumpkin seed crude protein with alkaline protease and trypsin. Compared with other enzymatic hydrolysis methods, the ability to scavenge peroxide free radicals is improved; compared with before enzymatic hydrolysis, arginine is enriched, the arginine content is increased, and arginine is the only substrate for endothelial nitric oxide synthase to produce vasodilator factor NO. In basic theory and clinical research, arginine supplements are used to treat certain cardiovascular diseases. The form of the peptide in the pumpkin seed protein peptide meets the characteristics of high-activity antihypertensive active peptides. Therefore, the pumpkin seed protein peptide prepared by the method of enzymatic hydrolysis of pumpkin seed crude protein with a composite protease has a good improvement effect on vascular endothelial cell dysfunction and can promote the generation of NO factor. The technical problem of the lack of bioactive peptides that can be used to improve vascular endothelial cell dysfunction in the prior art is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The antioxidant capacity index of pumpkin seed protein peptides with different treatments provided in Experiment 2 of the present invention;

[0026] Figure 2 This is a distribution diagram of peptide segment lengths in the pumpkin seed protein peptide for improving vascular endothelial cell dysfunction prepared in Example 1 provided in Experiment 4 of the present invention. DETAILED DESCRIPTION

[0027] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0028] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0029] On one hand, the present invention provides a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction, wherein the pumpkin seed protein peptide is obtained by enzymatic hydrolysis of pumpkin seed crude protein by a composite protease;

[0030] The composite protease comprises alkaline protease and trypsin;

[0031] The mass ratio of the alkaline protease to trypsin is 1:3 to 3:1.

[0032] Experiments have shown that the preparation of pumpkin seed protein peptides by enzymatic hydrolysis of pumpkin seed crude protein with alkaline protease and trypsin has improved the ability to scavenge peroxide free radicals compared with other enzymatic hydrolysis methods; compared with before enzymatic hydrolysis, arginine is enriched, the arginine content is increased, and arginine is the only substrate for endothelial nitric oxide synthase to produce vasodilator factor NO. In basic theory and clinical research, arginine supplements are used to treat certain cardiovascular diseases. The form of the peptide in the pumpkin seed protein peptide meets the characteristics of high-activity antihypertensive active peptides. Therefore, the pumpkin seed protein peptide prepared by the method of enzymatic hydrolysis of pumpkin seed crude protein with a composite protease has a good improvement effect on vascular endothelial cell dysfunction and can promote the generation of NO factor. The technical problem of the lack of drugs that can improve vascular endothelial cell dysfunction in the prior art is solved.

[0033] The mass ratio of alkaline protease to trypsin may be, but is not limited to, 1:3, 1.5:3, 2:3, 1:1, 1.5:1, 2:1, 2.5:1 or 3:1, or any ratio between 1:3 and 3:1.

[0034] In some specific embodiments, the mass ratio of alkaline protease to trypsin is 1:2 to 2:1.

[0035] In some specific embodiments, the added amount of the composite protease is 0.2-1.1% of the total mass of pumpkin seed crude protein.

[0036] The added amount of the composite protease can be, but is not limited to, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or 1.1% of the total mass of pumpkin seed crude protein.

[0037] According to another aspect of the present invention, there is also provided a method for preparing pumpkin seed protein peptide for improving vascular endothelial cell dysfunction, comprising the following steps:

[0038] A. Adding compound protease to pumpkin seed crude protein homogenate for enzymolysis;

[0039] B. The enzymatic hydrolysate prepared in step A is heated to inactivate the enzyme, the supernatant is obtained by centrifugation, and the supernatant is concentrated and dried to obtain pumpkin seed protein peptide.

[0040] In some specific embodiments, the method for preparing the pumpkin seed crude protein homogenate comprises adding water to the pumpkin seed crude protein and homogenizing to obtain the pumpkin seed crude protein homogenate.

[0041] In some specific embodiments, the mass ratio of pumpkin seed crude protein to water is 1:7.33-15.67.

[0042] Among them, the mass ratio of pumpkin seed crude protein to water can be but not limited to 1:7.33, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15 or 1:15.67, or it can be any ratio between 1:7.33 and 15.67.

[0043] In some specific embodiments, the enzymatic hydrolysis pH of the pumpkin seed crude protein homogenate is 7.5-10.

[0044] The enzymatic hydrolysis pH of the pumpkin seed crude protein homogenate can be, but is not limited to, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10, and can be any value between 7.5 and 10.

[0045] Specifically, the pH can be adjusted using sodium hydroxide or potassium hydroxide solutions of different concentrations.

[0046] In some specific embodiments, the enzymatic hydrolysis temperature is 48-57°C.

[0047] The temperature of the enzymatic hydrolysis may be, but is not limited to, 48°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C or 57°C, or any temperature between 48 and 57°C.

[0048] In some specific embodiments, the enzymatic hydrolysis time is 3 to 6 hours.

[0049] The enzymatic hydrolysis time may be, but is not limited to, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h or 6 h, and may be any time between 3 and 6 h.

[0050] According to another aspect of the present invention, there is also provided use of the pumpkin seed protein peptide or the pumpkin seed protein peptide prepared by the above preparation method in preparing a product for preventing or treating vascular endothelial cell dysfunction.

[0051] Among them, endothelial cell dysfunction includes impaired vasomotor function, proinflammatory state, thrombosis, oxidative stress and arterial wall proliferation caused by reduced NO production or utilization, vascular permeability disorder or increased ROS generation.

[0052] Oxidative stress is a classic mechanism that induces endothelial cell dysfunction. Under oxidative stress, ROS (reactive oxygen species) accumulate in endothelial cells. A large amount of ROS acts on the basement membrane of endothelial cells, damaging the body's antioxidant system and reducing the activity of antioxidant enzymes. Oxidative lipids increase the permeability of endothelial cells, causing active substances in blood vessels to invade endothelial cells and deposit in the basement membrane, damaging the structure and function of endothelial cells. ROS can activate signal molecules in the pro-inflammatory signaling pathway to promote the expression of inflammatory cytokines, leading to inflammatory reactions and endothelial cell apoptosis and shedding. At the same time, ROS inhibits the level of nitric oxide synthase (eNOS) and reduces the content of nitric oxide (NO). Nitric oxide deficiency further leads to endothelial dysfunction.

[0053] The present invention uses alkaline protease and trypsin to enzymatically hydrolyze pumpkin seed crude protein to prepare pumpkin seed protein peptide, which has a strong peroxide free radical scavenging ability, can remove ROS (reactive oxygen) in cells, reduce oxidative stress reaction, and has an improving effect on vascular endothelial cell dysfunction caused by oxidative stress, can significantly increase NO generation, and reduce the level of inflammatory factors. It is aimed at the mechanism of vascular endothelial cell dysfunction and fundamentally solves the problem.

[0054] Among them, the products include drugs or foods, or other products used to prepare the prevention or treatment of vascular endothelial cell dysfunction.

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] The pumpkin seed crude protein used in the following examples comes from Baoderui (Hubei) Health Industry Co., Ltd., and has a protein content of about 60%.

[0057] Example 1

[0058] 1) Add 1 kg of pumpkin seed crude protein into 9 kg of aqueous solution, stir evenly and then homogenize to obtain a homogenate.

[0059] 2) The pH value of the homogenate was adjusted to 8.5±0.5 with 5 mol / l sodium hydroxide, and then 4.5 g alkaline protease and 4.5 g trypsin were added, and the mixture was enzymolyzed at 52±2°C for 5 h to obtain an enzymolysis solution.

[0060] 3) The enzymatic hydrolysate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0061] Example 2

[0062] 1) Add 1 kg of pumpkin seed crude protein to 15.67 kg of aqueous solution, stir evenly and homogenize to obtain a homogenate.

[0063] 2) The pH value of the homogenate was adjusted to 8.0±0.5 with 5 mol / l sodium hydroxide, and then 2.75 g of alkaline protease and 8.25 g of trypsin were added, and the mixture was enzymolyzed at 50±2°C for 3 h to obtain an enzymolysis solution.

[0064] 3) The enzymatic hydrolysate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptides that improve vascular endothelial cell dysfunction.

[0065] Example 3

[0066] 1) Add 1 kg of pumpkin seed crude protein to 7.33 kg of aqueous solution, stir evenly and homogenize to obtain a homogenate.

[0067] 2) The pH value of the homogenate was adjusted to 9.0±0.5 with 5 mol / l sodium hydroxide, and then 1.5 g alkaline protease and 0.5 g trypsin were added, and the mixture was enzymolyzed at 52±2°C for 6 h to obtain an enzymolysis solution.

[0068] 3) The enzymatic hydrolysate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 10000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0069] Example 4

[0070] 1) Add 1 kg of pumpkin seed crude protein into 11.5 kg of aqueous solution, stir evenly and homogenize to obtain a homogenate.

[0071] 2) The pH value of the homogenate was adjusted to 9.5±0.5 with 5 mol / l sodium hydroxide, and then 3.33 g of alkaline protease and 1.67 g of trypsin were added, and the mixture was hydrolyzed at 55±2°C for 4 h to obtain an enzymatic solution.

[0072] 3) The enzymatic hydrolysate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0073] Example 5

[0074] 1) Add 1 kg of pumpkin seed crude protein into 9 kg of aqueous solution, stir evenly and then homogenize to obtain a homogenate.

[0075] 2) The pH value of the homogenate was adjusted to 8.0±0.5 with 5 mol / l sodium hydroxide, and then 2.33 g of alkaline protease and 4.67 g of trypsin were added, and the mixture was enzymolyzed at 52±2°C for 5 h to obtain an enzymolysis solution.

[0076] 3) The enzymatic hydrolysate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0077] Comparative Example 1

[0078] 1) Add 1 kg of pumpkin seed crude protein into 9 kg of aqueous solution, stir evenly and then homogenize to obtain a homogenate.

[0079] 2) The pH value of the homogenate was adjusted to 8.5±0.5 with 5 mol / l sodium hydroxide, and then 9 g of alkaline protease was added. The homogenate was hydrolyzed at the optimum temperature of 52±2°C for 5 h to obtain an enzymatic solution.

[0080] 3) The enzymatic hydrolyzate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptide.

[0081] Comparative Example 2

[0082] 1) Add 1 kg of pumpkin seed crude protein into 9 kg of aqueous solution, stir evenly and then homogenize to obtain a homogenate.

[0083] 2) The pH value of the homogenate was adjusted to 7.0±0.5 with 5 mol / l sodium hydroxide, and then 9 g of neutral protease was added. The homogenate was enzymolyzed at the optimal temperature of neutral protease, 50±2°C, for 5 h to obtain an enzymolysis solution.

[0084] 3) The enzymatic hydrolyzate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptide.

[0085] Comparative Example 3

[0086] 1) Add 1 kg of pumpkin seed crude protein into 9 kg of aqueous solution, stir evenly and then homogenize to obtain a homogenate.

[0087] 2) The pH value of the homogenate was adjusted to 8.5±0.5 with 5 mol / l sodium hydroxide, and then 9 g of trypsin was added. The homogenate was hydrolyzed at the optimum temperature of the enzyme, 52±2°C, for 5 h to obtain an enzymatic solution.

[0088] 3) The enzymatic hydrolyzate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptide.

[0089] Comparative Example 4

[0090] 1) Add 1 kg of pumpkin seed crude protein into 9 kg of aqueous solution, stir evenly and then homogenize to obtain a homogenate.

[0091] 2) The pH value of the homogenate was adjusted to 8.5±0.5 with 5 mol / l sodium hydroxide, and then 4.5 g of alkaline protease and 4.5 g of neutral protease were added, and the homogenate was enzymolyzed at 52±2°C for 5 h to obtain an enzymolysis solution.

[0092] 3) The enzymatic hydrolyzate was heated to 100°C and maintained for 30 minutes to inactivate the enzyme. After inactivation of the enzyme, the solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected, concentrated, and dried to obtain pumpkin seed protein peptide.

[0093] Experiment 1 Effects of different treatments on relative NO content

[0094] Eahy926 cells (human umbilical vein endothelial cells, purchased from Wuhan Punosai Life Science Co., Ltd.) were plated in 6-well plates and cultured with Eahy926 special culture medium (DMEM culture medium + 10% FBS + 1% double antibody) for 24 hours, and the cell culture medium was aspirated. Culture medium (normal group), hydrogen peroxide solution (model group, culture medium concentration of 6mmol / L), test sample solution (the sample is the peptide sample prepared in Example 1-9 and Comparative Example 1-6, and the sample solution is a mixed solution prepared with the sample and the hydrogen peroxide modeling agent, wherein the sample concentration is 8mg / mL and the hydrogen peroxide concentration is 6mmol / L), and positive drug solution (a mixed solution prepared with captopril and a hydrogen peroxide modeling agent, wherein the captopril concentration is 2μg / mL and the hydrogen peroxide concentration is 6mmol / L) were added to each space. After 24 hours of sample administration, the solution was aspirated and 200ul was used. PBS was used for washing to remove dead cells and residual sample solution, and then 200ul of trypsin was used for digestion. 600ul of culture medium was used to stop digestion, and the suspension containing trypsin and culture medium was transferred to a 1.5ml centrifuge tube, centrifuged at 1000r for 5 minutes, and the supernatant was discarded. 200ul of PBS solution was added to each well to wash the cells, and centrifuged at 1500r / min for 5 minutes. The centrifugation was repeated twice with PBS washing, and the supernatant PBS solution was discarded. 55ul of Western and IP cell lysis solution (P0013) was added to each tube of the cell pellet, and the cells were evenly washed and lysed on ice for 50 minutes. The centrifuge tube was then placed at 1500r for centrifugation for 10 minutes, and the supernatant was taken to detect the NO content using the Bio-Tech NO Kit (Cat. No.: S0021S). The relative NO content of the sample was calculated according to the following formula:

[0095] Relative NO content of sample = NO content of sample / NO content of normal group * 100%

[0096] The results are shown in Table 1.

[0097] Table 1

[0098] Group NO relative content % (m±SD) Normal group 100±8.80** Model Group 52.67±7.57 Captopril 88.85±11.77** Example 1 108.05±4.79***&& Example 2 106.84±3.39***&& Example 3 88.27±7.76** Example 4 104.25±17.83**& Example 5 111.73±10.57***&& Comparative Example 1 79.71±6.82* Comparative Example 2 59.17±10.44 Comparative Example 3 64.17±13.25 Comparative Example 4 71.25±14.74

[0099] Note: * indicates the sample group relative to the model group, & indicates the sample group relative to comparative example 1; one symbol represents P < 0.05, two identical symbols represent P < 0.01, and three identical symbols represent P < 0.001.

[0100] As shown in Table 1, the normal group and the model group have extremely significant differences, indicating that the modeling is successful, that is, peroxide causes dysfunction of human umbilical vein vascular endothelial cells, resulting in an extremely significant reduction in the amount of NO generated. The NO generation in Examples 1-5 is extremely significantly increased relative to the model group, indicating that the use of alkaline protease combined with trypsin to prepare pumpkin seed protein peptides has a good improvement in vascular endothelial cell dysfunction and can significantly promote the generation of NO factors. The generation of NO factors in Examples 1-5 relative to the model group increased by 67.59% to 112.13%. The production of NO in Comparative Example 1 is significantly increased relative to the model group, indicating that the preparation of pumpkin seed protein peptides by alkaline protease has the effect of improving vascular endothelial cell dysfunction, but the improvement is relatively weak, only increasing by 47.75%. The NO generation in Comparative Examples 2, 3, and 4 is slightly increased relative to the model group, but there is no significant difference, indicating that the pumpkin seed protein peptide prepared by neutral protease, trypsin, alkaline protease and neutral protease is not effective in improving vascular endothelial cell dysfunction.

[0101] The NO generation amount of Example 1 is significantly increased relative to that of Comparative Examples 1, 2, 3, and 4. This indicates that Example 1 has a better effect on improving vascular endothelial cell dysfunction relative to the comparative examples. The relative NO content generated by Example 1 relative to Comparative Examples 1, 2, 3, and 4 is increased by 38.85%, 82.61%, 68.38%, and 51.65%, respectively. Example 1 and Comparative Examples 1, 2, 3, and 4 are enzymolyzed under the same conditions (substrate concentration, enzyme addition amount, enzymolysis time), but the types of enzymes used for enzymolysis are different, which fully demonstrates that the pumpkin seed protein peptide prepared by enzymolysis of pumpkin seed protein by alkaline protease combined with pancreatic enzyme has a better effect on improving vascular endothelial cell dysfunction, while alkaline protease, pancreatic enzyme, neutral protease, or alkaline protease combined with neutral protease alone cannot achieve this effect.

[0102] Experiment 2 Effects of different treatments on the ability of pumpkin seed protein peptide to scavenge peroxide free radicals

[0103] In this experiment, the antioxidant capacity index (ORAC) in the publicly published "Principle and Application of Antioxidant Capacity Index (ORAC) Determination" (Xu Jiekun, Yao Xinsheng, Kurihara Hiroshi, "Chinese Pharmacological Bulletin", 2006(08):1015-1021) was used to evaluate the ability of pumpkin seed protein peptides prepared in Examples 1-5 and Comparative Examples 1-4 to scavenge peroxide free radicals.

[0104] Specific results such as Figure 1As shown. Examples 1-5 have very strong peroxide free radical scavenging ability, and the peroxide free radical scavenging ability activity values ​​are 7.34, 6.36, 6.07, 8.29, and 7.6, respectively; significantly higher than each comparative example. Among them, taking Example 1 as an example, the ability to scavenging peroxide free radicals is increased by 36.19%, 102.05%, 104.49%, and 50.11% relative to Comparative Examples 1, 2, 3, and 4, respectively.

[0105] Experiment 3 Effects of different treatments on the amino acid composition and structural components of pumpkin seed protein peptide

[0106] This test uses the method for determining amino acids in food in GB 5009.124 National Food Safety Standard to determine the amino acids in the pumpkin seed protein peptides and pumpkin seed crude protein raw materials of Examples 1 to 5. The determination results are shown in Table 2.

[0107] Table 2

[0108]

[0109] Among them, because the total amino acid content of each sample is different, in order to compare and analyze the composition structure of 16 kinds of amino acids at the same level, the relative amino acid percentage content is used, that is, amino acid content / total amount of 16 amino acids*100%.

[0110] As shown in Table 2, the amino acid composition of peptides obtained by enzymolysis changes relative to pumpkin seed crude protein. The basic amino acids in Examples 1-5 are enriched, and the basic amino acids are increased by 16.15% to 21.45% relative to pumpkin seed crude protein, among which arginine is greatly increased, from 13.99% of the raw material to 15.85% to 16.42%, an increase of more than 13.3%. L-arginine is the only substrate for endothelial nitric oxide synthase to produce vasodilator factor NO (NO is the most important factor affecting the dysfunction of vascular endothelial cells, regulating the homeostasis of the vascular environment, inhibiting platelet aggregation and preventing thrombosis). In basic theory and clinical research, L-arginine supplements are used to treat certain cardiovascular diseases such as hypertension and coronary heart disease. The content of acidic amino acids also increased by 11.74%, while the content of non-polar amino acids decreased, decreasing by more than 20.19%.

[0111] Experiment 4 Effects of different treatments on the peptide composition of pumpkin seed protein peptides

[0112] The pumpkin seed protein peptide prepared in Example 1 was analyzed by LC-MS / MS equipped with an online nanospray ion source. The whole system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with EASY-nanoLC 1200. A total of 2 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient of 60 min. The column flow was controlled at 300 nL / min, the column temperature was 40 ° C, the electrospray voltage was 2 kV, and the gradient started from 4% of phase B, increased to 50% in 53.6 minutes with a nonlinear gradient, increased to 95% in 40 seconds, and maintained for 5.6 minutes.

[0113] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 350-1500; resolution: 120000; Normalized AGC target: 200%; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15000; Normalized AGC target: 100%; maximum injection time: 25 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s.

[0114] Tandem mass spectra were analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc, Waterloo, Canada). The database was NCBI-Cucurbita moschata (version 2023, 44191 entries) database, set to none. The search parameters were fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, variable modification: Oxidation (M) 15.99, Deamidation (NQ) 0.98. The protein card value was at least 1 unique peptide; the peptide card value was -10lgP ≥ 20. If a new peptide or an unknown modified peptide was found, that is, no matching peptide was retrieved in the MS / MS spectrum database, but the De Novo detection of the spectrum was highly reliable, the denovo score of the included peptides needed to be above 98.

[0115] The experimental results are shown in Tables 3 to 5.

[0116] Table 3 Information of main peptide molecules in pumpkin seed protein peptide prepared in Example 1 (1-84)

[0117]

[0118]

[0119] Table 4 Information of main peptide molecules in pumpkin seed protein peptide prepared in Example 1 (85-168)

[0120]

[0121] Table 5 Information of main peptide molecules in pumpkin seed protein peptide prepared in Example 1 (169-277)

[0122]

[0123] It can be seen that the peptides in pumpkin seed protein peptides are mainly composed of 3 to 10 peptides, accounting for about 81.94%. The specific peptide length distribution is as follows Figure 2 As shown. The molecular weight of the peptides is distributed in a gradient, with small molecule peptides within 1000 accounting for 55.95%, peptides with a molecular weight of 1000 to 3000 accounting for 41.41%, and peptides above 3000 accounting for 2.64%. From the perspective of amino acid composition, the C-terminal hydrophobic amino acids account for 55.95%, the C-terminal branched amino acid peptides account for 37% of the total peptides, and the C-terminal hydrophobic amino acid peptides account for 66.14%, and are mainly leucine. Peptides containing arginine account for 39.65%, of which arginine at the C-terminal accounts for 56.67%.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pumpkin seed protein peptide for improving vascular endothelial cell dysfunction, characterized in that: The pumpkin seed protein peptide is obtained by enzymolysis of pumpkin seed crude protein with a composite protease; The composite protease comprises alkaline protease and trypsin; The mass ratio of the alkaline protease to trypsin is 1:3 to 3:

1.

2. The pumpkin seed protein peptide according to claim 1, characterized in that The mass ratio of the alkaline protease to trypsin is 1:2 to 2:

1.

3. The pumpkin seed protein peptide according to claim 2, characterized in that The added amount of the composite protease is 0.2-1.1% of the crude protein mass of pumpkin seeds.

4. The method for preparing a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction according to any one of claims 1 to 3, characterized in that: The following steps are involved: A. Adding compound protease to pumpkin seed crude protein homogenate for enzymolysis; B. The enzymatic hydrolysate prepared in step A is heated to inactivate the enzyme, the supernatant is obtained by centrifugation, and the supernatant is concentrated and dried to obtain pumpkin seed protein peptide.

5. The preparation method according to claim 4, characterized in that: The method for preparing the pumpkin seed crude protein homogenate comprises adding water to the pumpkin seed crude protein and homogenizing to obtain the pumpkin seed crude protein homogenate.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the pumpkin seed crude protein to water is 1:7.33-15.

67.

7. The preparation method according to claim 5, characterized in that: The enzymatic hydrolysis pH of the pumpkin seed crude protein homogenate is 7.5-10.

8. The preparation method according to claim 4, characterized in that: The temperature of the enzymatic hydrolysis is 48-57°C.

9. The preparation method according to claim 8, characterized in that: The enzymatic hydrolysis time is 3 to 6 hours.

10. Use of the pumpkin seed protein peptide according to any one of claims 1 to 3 or the pumpkin seed protein peptide prepared by the preparation method according to any one of claims 4 to 9 in preparing a product for preventing or treating vascular endothelial cell dysfunction.

Images