A pumpkin seed protein peptide for improving vascular endothelial cell dysfunction and its preparation method

Abstract

The present invention provides a pumpkin seed protein peptide for improving vascular endothelial cell dysfunction and a preparation method thereof, relating to the technical field of bioactive peptides. The pumpkin seed protein peptide is obtained by enzymatically hydrolyzing pumpkin seed crude protein with a compound protease. The compound protease includes alkaline protease and trypsin. The mass ratio of alkaline protease to trypsin is 1:3 to 3:1. The pumpkin seed protein peptide prepared by enzymatically hydrolyzing pumpkin seed crude protein with alkaline protease in combination with trypsin has improved peroxide radical scavenging ability compared with other enzymatic hydrolysis methods, and arginine is enriched. The form of the peptide in the pumpkin seed protein peptide conforms to the characteristics of highly active antihypertensive active peptides, and has a good effect on improving vascular endothelial cell dysfunction and can promote the generation of NO factor. It solves the technical problem in the prior art of the lack of bioactive peptides that can be used to improve vascular endothelial cell dysfunction.

Description

Technical Field

[0001] The present 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 (endothelial cells) serve as a mechanical barrier between vascular smooth muscle and blood and are the target organ for many active substances. They secrete vasodilators and vasoconstrictors, the balance of which regulates vasomotion, promotes the balance between the fibrinolytic system and coagulation, inhibits platelet aggregation and inflammatory cell adhesion to the endothelium, and regulates vascular smooth muscle growth. Endothelial dysfunction manifests as decreased nitric oxide (NO) production or utilization, disrupted vascular permeability, and increased ROS production, ultimately leading to adverse consequences such as impaired vasomotor function, a proinflammatory state, thrombosis, oxidative stress, and arterial wall proliferation.

[0003] Research has shown that endothelial cell dysfunction is a key factor in the development of diseases such as hypertension, diabetes, atherosclerosis, and heart failure. Improving endothelial dysfunction plays an important role in preventing and treating cardiovascular diseases. However, there are currently very few drugs that specifically improve 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 in the prior art of lacking bioactive peptides that can be used to improve vascular endothelial cell dysfunction.

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

[0007] A 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-mentioned preparation method in the preparation of a drug 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 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 with 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 the alkaline protease to the trypsin is 1:2 to 2:1.

[0013] Furthermore, the addition amount of the compound protease is 0.2 to 1.1% of the total mass of the crude pumpkin seed protein.

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

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

[0016] B. Heating the enzymatic hydrolysate prepared in step A to inactivate the enzyme, centrifuging to obtain the supernatant, and concentrating and drying to obtain pumpkin seed protein peptide.

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

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

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

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

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

[0022] In a third aspect, the present invention provides the application 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 products for preventing or treating vascular endothelial cell dysfunction.

[0023] The pumpkin seed protein peptide for improving vascular endothelial cell dysfunction provided by the present invention uses alkaline protease combined with trypsin to enzymatically hydrolyze crude pumpkin seed protein to prepare pumpkin seed protein peptide. Compared with other enzymatic hydrolysis methods, it improves the ability to scavenge peroxide free radicals; compared with before enzymatic hydrolysis, arginine is enriched and the arginine content is increased, and arginine is the only substrate for endothelial nitric oxide synthase to produce the vasodilator NO. Arginine supplements are used to treat certain cardiovascular diseases in basic theory and clinical research. The form of the peptide in the pumpkin seed protein peptide conforms to the characteristics of highly active antihypertensive active peptides. Therefore, the pumpkin seed protein peptide prepared by enzymatically hydrolyzing crude pumpkin seed protein with compound protease has a good improvement effect on vascular endothelial cell dysfunction and can promote the generation of NO factor. It solves the technical problem in the prior art of lacking bioactive peptides that can be used to improve vascular endothelial cell dysfunction. Description of the Drawings

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

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

[0026] Figure 2 Peptide length distribution diagram of the pumpkin seed protein peptide prepared in Example 1 for improving vascular endothelial cell dysfunction provided for Experiment 4 of the present invention. Specific embodiments

[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 meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0028] Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

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

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

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

[0032] Experimental results show that when alkaline protease and trypsin are used in combination to hydrolyze crude pumpkin seed protein to prepare pumpkin seed protein peptides, compared with other hydrolysis methods, the ability to scavenge peroxyl radicals is improved; compared with before hydrolysis, arginine is enriched and its content is increased. Arginine is the only substrate for endothelial nitric oxide synthase to produce the vasodilator NO, and arginine supplements are used to treat certain cardiovascular diseases in basic theory and clinical research. The form of the peptides in pumpkin seed protein peptides conforms to the characteristics of highly active antihypertensive peptides. Therefore, the pumpkin seed protein peptides prepared by hydrolyzing crude pumpkin seed protein with a combination of proteases have a good effect on improving vascular endothelial cell dysfunction and can promote the production of NO factor. This solves the technical problem in the prior art of the lack of drugs capable of improving vascular endothelial cell dysfunction.

[0033] Among them, the mass ratio of alkaline protease to trypsin can 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, and can also be any ratio between 1:3 and 3:1.

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

[0035] In some specific embodiments, the addition amount of the combination of proteases is 0.2 to 1.1% of the total mass of the crude pumpkin seed protein.

[0036] Among them, the addition amount of the combination of proteases 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 the crude pumpkin seed protein.

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

[0038] A. Adding a combination of proteases to the crude pumpkin seed protein homogenate for hydrolysis;

[0039] B. Heating the hydrolyzate prepared in step A to inactivate the enzyme, centrifuging to obtain the supernatant, and concentrating and drying to obtain pumpkin seed protein peptides.

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

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

[0042] Among them, the mass ratio of crude pumpkin seed protein to water can be, but is 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, and can also be any ratio between 1:7.33 and 15.67.

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

[0044] Among them, the pH of enzymatic hydrolysis of the crude pumpkin seed protein homogeneous slurry 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 also be any value between 7.5 and 10.

[0045] Specifically, sodium hydroxide or potassium hydroxide solutions with different concentrations can be used to adjust the pH.

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

[0047] Among them, the temperature of enzymatic hydrolysis can 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, and can also be any temperature between 48 and 57 °C.

[0048] In some specific embodiments, the time of enzymatic hydrolysis is 3 - 6 h.

[0049] Among them, the time of enzymatic hydrolysis can 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 can also be any time between 3 and 6 h.

[0050] According to another aspect of the present invention, there is also provided 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 products for preventing or treating vascular endothelial cell dysfunction.

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

[0052] Oxidative stress is a classic mechanism that induces vascular endothelial cell dysfunction. Under oxidative stress stimulation, the accumulation of ROS (reactive oxygen species) in endothelial cells occurs. A large amount of ROS acts on the basement membrane of vascular endothelial cells, damaging the body's antioxidant system and reducing the activity of antioxidant enzymes. Oxidizing the lipids therein increases the permeability of vascular endothelial cells, leading to the invasion of active substances in the blood vessels into endothelial cells and deposition in the basement membrane, damaging the structure and function of vascular endothelial cells. ROS can activate signal molecules in the pro-inflammatory signaling pathway to promote the expression of inflammatory cytokines, leading to inflammatory responses and apoptosis and shedding of endothelial cells. At the same time, ROS inhibits the level of nitric oxide synthase (eNOS) and reduces the content of nitric oxide (NO). The lack of nitric oxide further leads to endothelial dysfunction.

[0053] The present invention uses alkaline protease in combination with trypsin to enzymatically hydrolyze crude pumpkin seed protein to prepare pumpkin seed protein peptides, which have strong peroxyl radical scavenging ability, can scavenge ROS (reactive oxygen species) in cells, and reduce oxidative stress responses. It has an improving effect on vascular endothelial cell dysfunction caused by oxidative stress, can significantly increase the production of NO, and reduce the level of inflammatory factors. It addresses the problem at the root in view of the mechanism of vascular endothelial cell dysfunction.

[0054] Among them, the product includes a drug or food, or other products for preparing a product for preventing or treating vascular endothelial cell dysfunction.

[0055] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

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

[0057] Example 1

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

[0059] 2) Adjust the pH value of the homogeneous slurry to 8.5 ± 0.5 with 5 mol / l sodium hydroxide, then add 4.5 g of alkaline protease and 4.5 g of trypsin, and enzymatically hydrolyze for 5 h at a temperature of 52 ± 2 °C to obtain an enzymatic hydrolysate.

[0060] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 8000 r / min for 10 min, collect the supernatant, concentrate and dry it to obtain pumpkin seed protein peptides for improving vascular endothelial dysfunction.

[0061] Example 2

[0062] 1) Add 1 kg of crude pumpkin seed protein to 15.67 kg of aqueous solution, stir evenly and then perform homogenization treatment to obtain a homogeneous slurry.

[0063] 2) Adjust the pH value of the homogeneous slurry to 8.0 ± 0.5 with 5 mol / l sodium hydroxide, then add 2.75 g of alkaline protease and 8.25 g of trypsin, and enzymatically hydrolyze for 3 h at a temperature of 50 ± 2 °C to obtain an enzymatic hydrolysate.

[0064] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 8000 r / min for 10 min, collect the supernatant, concentrate and dry it to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0065] Example 3

[0066] 1) Add 1 kg of crude pumpkin seed protein to 7.33 kg of aqueous solution, stir evenly and then perform homogenization treatment to obtain a homogeneous slurry.

[0067] 2) Adjust the pH value of the homogeneous slurry to 9.0 ± 0.5 with 5 mol / l sodium hydroxide, then add 1.5 g of alkaline protease and 0.5 g of trypsin, and enzymatically hydrolyze for 6 h at a temperature of 52 ± 2 °C to obtain an enzymatic hydrolysate.

[0068] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 10000 r / min for 10 min, collect the supernatant, concentrate and dry it to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0069] Example 4

[0070] 1) Add 1 kg of crude pumpkin seed protein to 11.5 kg of aqueous solution, stir evenly and then perform homogenization treatment to obtain a homogeneous slurry.

[0071] 2) Adjust the pH value of the homogeneous slurry to 9.5 ± 0.5 with 5 mol / l sodium hydroxide, then add 3.33 g of alkaline protease and 1.67 g of trypsin, and enzymatically hydrolyze for 4 h at a temperature of 55 ± 2 °C to obtain an enzymatic hydrolysate.

[0072] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 8000 r / min for 10 min, collect the supernatant, concentrate and dry it to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0073] Example 5

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

[0075] 2) Adjust the pH value of the homogeneous slurry to 8.0 ± 0.5 with 5 mol / l sodium hydroxide, then add 2.33 g of alkaline protease and 4.67 g of trypsin, and enzymatically hydrolyze at a temperature of 52 ± 2 °C for 5 h to obtain an enzymatic hydrolysate.

[0076] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 8000 r / min for 10 min, collect the supernatant and concentrate and dry it to obtain pumpkin seed protein peptides that improve vascular endothelial dysfunction.

[0077] Comparative Example 1

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

[0079] 2) Adjust the pH value of the homogeneous slurry to 8.5 ± 0.5 with 5 mol / l sodium hydroxide, then add 9 g of alkaline protease, and enzymatically hydrolyze at the optimal temperature of the enzyme 52 ± 2 °C for 5 h to obtain an enzymatic hydrolysate.

[0080] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 8000 r / min for 10 min, collect the supernatant and concentrate and dry it to obtain pumpkin seed protein peptides.

[0081] Comparative Example 2

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

[0083] 2) Adjust the pH value of the homogeneous slurry to 7.0 ± 0.5 with 5 mol / l sodium hydroxide, then add 9 g of neutral protease, and enzymatically hydrolyze at the optimal temperature of neutral protease 50 ± 2 °C for 5 h to obtain an enzymatic hydrolysate.

[0084] 3) Heat the enzymatic hydrolysate to 100 °C and keep it for 30 min for enzyme inactivation treatment. After enzyme inactivation, centrifuge at 8000 r / min for 10 min, collect the supernatant and concentrate and dry it to obtain pumpkin seed protein peptides.

[0085] Comparative Example 3

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

[0087] 2) Adjust the pH value of the homogeneous slurry to 8.5 ± 0.5 with 5 mol / l sodium hydroxide, then add 9 g of trypsin, and enzymatically hydrolyze at the optimal temperature of the enzyme 52 ± 2 °C for 5 h to obtain an enzymatic hydrolysate.

[0088] 3) The enzymatic hydrolysate was heated to 100 °C and maintained for 30 min for inactivating enzymes. After enzyme inactivation, it was centrifuged at 8000 r / min for ......

[0089] Comparative Example 4

[0090] 1) 1 kg of crude pumpkin seed protein was added to 9 kg of aqueous solution, and after stirring evenly, it was homogenized to obtain a homogeneous slurry.

[0091] 2) The pH value of the homogeneous slurry 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. It was enzymatically hydrolyzed at a temperature of 52 ± 2 °C for 5 h to obtain an enzymatic hydrolysate.

[0092] 3) The enzymatic hydrolysate was heated to 100 °C and maintained for 30 min for inactivating enzymes. After enzyme inactivation, it was centrifuged at 8000 r / min for 10 min, and the supernatant was collected for concentration and drying to obtain pumpkin seed protein peptides.

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

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

[0095] Relative content of sample NO = Sample NO content / Normal group NO content * 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 compared with the model group, and & indicates the sample group compared with 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 can be seen from Table 1, there are extremely significant differences between the normal group and the model group, indicating that the model was successfully established, that is, peroxides caused dysfunction of human umbilical vein endothelial cells, resulting in an extremely significant decrease in the production of NO. The NO production in Examples 1-5 was extremely significantly increased compared with the model group, indicating that the preparation of pumpkin seed protein peptides using alkaline protease combined with trypsin had a good effect on improving endothelial cell dysfunction and could significantly promote the production of NO factor. The production of NO factor in Examples 1-5 increased by 67.59% to 112.13% compared with the model group. The production of NO in Comparative Example 1 was significantly increased compared with the model group, indicating that the preparation of pumpkin seed protein peptides using alkaline protease had the effect of improving endothelial cell dysfunction, but the improvement was relatively weak, only increasing by 47.75%. The production of NO in Comparative Examples 2, 3, and 4 increased slightly compared with the model group, but there was no significant difference, indicating that the pumpkin seed protein peptides prepared using neutral protease, trypsin, or alkaline protease combined with neutral protease had no obvious effect on improving endothelial cell dysfunction.

[0101] The NO production in Example 1 was extremely significantly increased compared with the groups of Comparative Examples 1, 2, 3, and 4. This indicates that Example 1 had a better effect on improving endothelial cell dysfunction compared with the comparative examples. The relative content of NO produced in Example 1 increased by 38.85%, 82.61%, 68.38%, and 51.65% respectively compared with Comparative Examples 1, 2, 3, and 4. Example 1 and Comparative Examples 1, 2, 3, and 4 were enzymatically hydrolyzed under the same conditions (substrate concentration, enzyme addition amount, enzymatic hydrolysis time), only the types of enzymes used for enzymatic hydrolysis were different. This fully shows that the pumpkin seed protein peptides prepared by enzymatically hydrolyzing pumpkin seeds with alkaline protease combined with trypsin have a good effect on improving endothelial cell dysfunction, while using alkaline protease, trypsin, neutral protease alone or alkaline protease combined with neutral protease cannot achieve this effect.

[0102] Experiment 2: Effect of different treatments on the ability of pumpkin seed protein peptides to scavenge peroxide radicals

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

[0104] The specific results are as Figure 1As shown. Examples 1 - 5 have very strong peroxide radical scavenging ability, and the vitality values of peroxide radical scavenging ability are 7.34, 6.36, 6.07, 8.29, and 7.6 respectively; significantly higher than those of each comparative example. Among them, taking Example 1 as an example, the ability to scavenge peroxide radicals increased by 36.19%, 102.05%, 104.49%, and 50.11% respectively compared with Comparative Examples 1, 2, 3, and 4.

[0105] Experiment 3 Effects of Different Treatments on the Amino Acid Composition and Structural Components of Pumpkin Seed Protein Peptides

[0106] In this experiment, the amino acids in the pumpkin seed protein peptides and pumpkin seed crude protein raw materials of Examples 1 - 5 were determined by the method for the determination of amino acids in foods in the national food safety standard GB 5009.124. The determination results are shown in Table 2.

[0107] Table 2

[0108]

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

[0110] As can be seen from Table 2, the amino acid composition of the peptides obtained by enzymatic hydrolysis changed relative to that of pumpkin seed crude protein. Basic amino acids were enriched in Examples 1 - 5, and the basic amino acids increased by 16.15% to 21.45% relative to pumpkin seed crude protein. Among them, arginine increased significantly, rising from 13.99% in the raw material to 15.85% to 16.42%, with an increase of more than 13.3%. L-arginine is the only substrate for the production of the vasodilator NO by endothelial nitric oxide synthase (NO is the most important factor affecting the dysfunction of vascular endothelial cells, regulating the homeostasis of the vascular internal environment, inhibiting platelet aggregation, and preventing thrombosis formation). 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, with a decrease of more than 20.19%.

[0111] Experiment 4 Effects of Different Treatments on the Peptide Composition Forms in Pumpkin Seed Protein Peptides

[0112] Analysis was performed using LC-MS. The pumpkin seed protein peptides prepared in Example 1 were analyzed by LC-MS / MS equipped with an online nano-spray ion source. The entire system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with EASY-nanoLC 1200. A total of 2 μL of the sample was loaded (analysis column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated with a 60-min gradient. The column flow rate 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 a non-linear gradient at 53.6 minutes, increased to 95% in 40 seconds, and was maintained for 5.6 minutes.

[0113] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 350 - 1500; resolution: 120,000; Normalized AGC target: 200%; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15,000; Normalized AGC target: 100%; maximum injection time: 25 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s.

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

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

[0116] Table 3 Information Table of Main Peptide Molecules in Pumpkin Seed Protein Peptides Prepared in Example 1 (1 - 84)

[0117]

[0118]

[0119] Table 4 Information Table of Main Peptide Molecules in Pumpkin Seed Protein Peptide Prepared in Example 1 (85 - 168)

[0120]

[0121] Table 5 Information Table 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 peptide are mainly composed of 3 - peptide to 10 - peptide, accounting for about 81.94%. The specific peptide length distribution is as Figure 2 shown. The molecular weights of the peptides are distributed in a gradient. The proportion of small - molecule peptide segments within 1000 is 55.95%, the proportion of peptide segments with molecular weights of 1000 - 3000 is 41.41%, and the proportion of those above 3000 is 2.64%. From the perspective of amino acid composition, the proportion of hydrophobic amino acids at the C - terminus is 55.95%. The proportion of peptide segments with branched - chain amino acids at the C - terminus accounts for 37% of the total peptide segments, and the proportion of peptide segments with hydrophobic amino acids at the C - terminus is 66.14%, and it is mainly leucine. The proportion of peptide segments containing arginine is 39.65%, and the proportion of arginine at the C - terminus in the peptide segments containing arginine is 56.67%.

[0124] Finally, it should be noted that the above - mentioned 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of pumpkin seed protein peptides for improving vascular endothelial cell dysfunction, characterized in that, It includes the following steps: A. Adding a compound protease to the crude pumpkin seed protein homogenate for enzymatic hydrolysis; B. Heating the enzymatic hydrolysate prepared in step A to inactivate the enzyme, centrifuging to obtain the supernatant, and concentrating and drying to obtain pumpkin seed protein peptides; The preparation method of the crude pumpkin seed protein homogenate includes adding water to the crude pumpkin seed protein and homogenizing to obtain the crude pumpkin seed protein homogenate; The mass ratio of the crude pumpkin seed protein to water is 1:7.33 to 15.67; The time for enzymatic hydrolysis is 3 to 6 hours; The compound protease includes alkaline protease and trypsin; The mass ratio of the alkaline protease to trypsin is 1:2 to 2:1; The addition amount of the compound protease is 0.2 to 1.1% of the mass of the crude pumpkin seed protein; The pH for enzymatic hydrolysis of the crude pumpkin seed protein homogenate is 7.5 to 10; The temperature for enzymatic hydrolysis is 48 to 57 °C.

2. Use of the pumpkin seed protein peptides prepared by the preparation method according to claim 1 in the preparation of a drug for preventing or treating vascular endothelial cell dysfunction.

Images