Antioxidant and anti-aging active peptides from pigeon blood and preparation method and application thereof

Abstract

The application discloses pigeon blood antioxidant and anti-aging active peptide and a preparation method and application thereof. The preparation method of the pigeon blood antioxidant and anti-aging active peptide comprises the following steps: adding an anticoagulant to pigeon blood and performing hemolysis pretreatment to dissolve pigeon blood hemoglobin; after the pretreatment, deionized water is added, the blood-water ratio and pH value are adjusted, a protease is added for enzymolysis, the enzyme is inactivated after the enzymolysis is completed, the temperature is cooled to room temperature, the pH value is adjusted to 7.0, and centrifugation is performed to obtain supernatant, and pigeon blood enzymolysis polypeptide is obtained after drying; and the protease is pepsin or trypsin. The pigeon blood active polypeptide prepared by the application can realize the antioxidant and anti-aging effects by adjusting the levels of oxidative stress, monoamine oxidase (MAO), advanced glycation end products (AGEs) and Klotho protein of an aging-regulating gene in brain tissues of D-galactose-induced aging mice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional peptide development, and particularly relates to pigeon blood antioxidant and anti-aging active peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Meat pigeons are rich in nutrients and are known as "one pigeon is better than nine chickens". The breeding quantity and consumption quantity thereof are increasing year by year. Pigeon blood is one of main by-products in the process of meat pigeon slaughtering and processing. Animal blood contains rich protein, which accounts for 10-20% of the content of whole blood, close to or higher than the protein content in muscle tissue of the same kind of livestock and poultry, and is called "liquid meat", which is a very important animal protein resource. However, at present, the pigeon blood in the process of meat pigeon slaughtering and processing is usually directly discharged as waste or used as animal feed, causing serious resource waste and environmental pollution. Modern medicine believes that the pigeon blood has the effects of strengthening body, beautifying skin, producing and supplementing blood, tonifying qi and prolonging life, and is a traditional tonic, which can be used as a high-quality raw material for the development of functional food and food-derived active peptide. At present, the research on animal blood at home and abroad mainly focuses on poultry chicken, duck blood and livestock pig, cow and deer blood, and the research on deep processing and functional activity of pigeon blood is relatively less.

[0003] In recent years, with the aggravation of population aging, various diseases related to aging, such as chronic diseases of cardiovascular and cerebrovascular diseases, neurodegenerative diseases and cancer, have a high incidence, which brings a series of health, social and medical problems. Population aging has become the primary problem faced by the world. How to delay aging and develop functional food with anti-aging effect has become a research hotspot in the field of life science.

[0004] In the theory of aging mechanism, the free radical theory is one of the most internationally recognized mechanisms. The aging process itself is caused by the imbalance between free radicals and antioxidants in the body, leading to oxidative stress, thus producing cell damage to accelerate aging, so that antioxidant and anti-aging are closely related. In recent years, it has been found that some polysaccharides (such as fungal polysaccharides, Chinese wolfberry polysaccharides and algal polysaccharides), unsaturated fatty acids (such as DHA and alpha-linolenic acid) and polypeptides (such as sea cucumber polypeptide, tilapia polypeptide and glutathione) have good antioxidant and anti-aging effects. Among them, polypeptides are concerned due to good solubility, easy absorption, low allergenicity and multiple physiological functions.

[0005] At present, there is no related report on the preparation of antioxidant and anti-aging active polypeptide from pigeon blood. SUMMARY

[0006] The present application aims to provide pigeon blood antioxidant and anti-aging active peptide as well as a preparation method and application thereof.

[0007] The object of the present application is achieved by the following technical solutions.

[0008] The preparation method of pigeon blood antioxidant and anti-aging active peptide comprises the following steps:

[0009] (1) The pigeon blood is added with an anticoagulant and subjected to hemolysis pretreatment to dissolve pigeon blood hemoglobin;

[0010] (2) After the pretreatment, deionized water is added to adjust the blood-water ratio and pH value, a protease is added for enzymolysis, and after the enzymolysis is completed, the enzyme is inactivated, and after being cooled to room temperature, the pH value is adjusted to 7.0, and the supernatant is obtained by centrifugation, and after drying, pigeon blood enzymolysis polypeptide is obtained, which is pigeon blood antioxidant and anti-aging active polypeptide.

[0011] The inventors found in research that the pigeon blood active polypeptide prepared by the above method has good antioxidant and anti-aging activity.

[0012] Further, in the above step (1), the anticoagulant is preferably 0.4% sodium citrate solution; the pretreatment conditions are: repeated freezing and thawing 1-3 times at -15 to -20℃, or homogenization by a high-speed shearing machine at 5000-8000 rpm for 3-5 min, or ultrasonic treatment by an ultrasonic cell disrupter at 20 kHz and 100-300 W for 3-5 min;

[0013] Most preferably, in step (1), the pretreatment conditions are: ultrasonic treatment by an ultrasonic cell disrupter at 20 kHz and 100 W for 5 min.

[0014] Further, in the above step (2), the blood-water ratio is the mass ratio of pigeon blood to deionized water of 1:0.5-1:2.5;

[0015] Further, in the above step (2), the protease is selected from pepsin or trypsin, preferably pepsin.

[0016] The inventors found in research that in the preparation method of the present application, the selection of the enzyme plays an important role in the antioxidant and anti-aging activity of the pigeon blood polypeptide prepared; research shows that the DPPH and ABTS free radical scavenging activity (antioxidant in vitro index) and tyrosine kinase inhibitory activity (anti-aging in vitro index) of the pigeon blood active polypeptide prepared by pepsin and trypsin hydrolysis are significantly higher than those of other proteases, but the protein recovery rate of the enzymolysis solution after trypsin hydrolysis is lower, therefore, the optimal hydrolysis protease is pepsin.

[0017] Further, in the above step (2), the pepsin enzymolysis conditions are: temperature 35-40℃, pH value 1.5-2.0, enzyme addition amount 4000-12000 U / g, and enzymolysis time 1-5 h;

[0018] Further, in the step (2), the trypsin enzymolysis conditions are: temperature 35-40℃, pH value 7.0-8.0, enzyme addition amount 4000-12000 U / g, and enzymolysis time 1-5 h.

[0019] Further, in the step (2), the enzyme inactivation conditions are: temperature 90-100℃, and time 10-15 min.

[0020] The application also claims the use of the pigeon blood antioxidant and anti-aging active peptide prepared by the above preparation method in anti-aging functional food, health products, cosmetics or medicines.

[0021] The application has the following advantages and effects relative to the prior art:

[0022] 1. The pigeon blood is used as a raw material, and a proteinase hydrolysis method is used to prepare an antioxidant and anti-aging active peptide. First, a pretreatment technology is used to improve the hemoglobin dissolution rate of pigeon blood. The protein recovery rate, the degree of hydrolysis, the DPPH and ABTS antioxidant activities, and the tyrosinase inhibitory activity of the pigeon blood enzymolysis liquid are evaluated, and pepsin and trypsin are selected as the preferred enzyme types for pigeon blood protein enzymolysis, and the pepsin is the best. Further, a single factor and orthogonal test are used to optimize the process of preparing the antioxidant and anti-aging polypeptide from pigeon blood enzymolysis, and the optimal hydrolysis process of pepsin is selected: the blood-water ratio is 1:1.2, the temperature is 37℃, the pH value is 2.0, the enzyme addition amount is 12000 U / g, and the enzymolysis time is 4 h. The pigeon blood active peptide obtained under the process mainly consists of peptide segments with a molecular weight less than 5000 kDa (accounting for 78.98%), has a high content of essential amino acids and antioxidant and anti-aging active amino acids, and reaches 42.01% and 27.22%, respectively.

[0023] 2. According to the animal experiment results, the pigeon blood active polypeptide prepared by the application can realize its antioxidant and anti-aging effects by regulating the oxidative stress in D-galactose-induced aging mice, the levels of monoamine oxidase (MAO), advanced glycation end products (AGEs) and the Klotho protein in brain tissue, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The hemoglobin dissolution rate of pigeon blood under different pretreatment methods.

[0025] Figure 2 The protein recovery rate, the degree of hydrolysis, the DPPH and ABTS free radical scavenging rate, and the tyrosinase inhibitory activity under different enzyme types.

[0026] Figure 3 The influence of the blood-water ratio on the pigeon blood enzymolysis effect.

[0027] Figure 4 Effect of enzyme addition amount on pigeon blood enzymolysis effect.

[0028] Figure 5 Effect of enzymeolysis time on pigeon blood enzymolysis effect.

[0029] Figure 6 Effect of pigeon blood active polypeptide on serum biochemical indexes of D-galactose-induced aging mice.

[0030] Figure 7 Effect of pigeon blood active polypeptide on brain tissue biochemical indexes of D-galactose-induced aging mice. DETAILED DESCRIPTION

[0031] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0032] In the application, the pigeon blood is taken from 21-day-old white-feathered Wang pigeons.

[0033] The pepsin and trypsin used are purchased from Sigma Company, USA.

[0034] Example 1

[0035] The preparation method of the pigeon blood antioxidant and anti-aging active peptide specifically comprises the following steps:

[0036] (1) The pigeon blood is added into 0.4% sodium citrate solution for anticoagulation, and is subjected to repeated freezing and thawing at -20℃ for 2 times to dissolve hemoglobin in the pigeon blood;

[0037] (2) After the pretreatment, a certain amount of deionized water is added to adjust the blood-water ratio to 1:1, the pH value is adjusted to 7.5, 4000 U / g of trypsin is added, and the mixture is placed in a constant-temperature water bath oscillator at 37℃ for 3 hours. After the enzymolysis is completed, the enzyme is inactivated by being placed at 95℃ for 10 minutes. After being cooled to room temperature, the pH value is adjusted to 7.0, and the mixture is centrifuged to obtain a supernatant. The supernatant is dried to obtain pigeon blood enzymolysis polypeptide, which is the pigeon blood antioxidant and anti-aging active polypeptide.

[0038] Example 2

[0039] The preparation method of the pigeon blood antioxidant and anti-aging active peptide specifically comprises the following steps:

[0040] (1) The pigeon blood is added into 0.4% sodium citrate solution for anticoagulation, and is subjected to repeated freezing and thawing at -20℃ for 2 times to dissolve hemoglobin in the pigeon blood;

[0041] (2) After the pretreatment, a certain amount of deionized water was added, the blood-water ratio was adjusted to 1:2, the pH value was adjusted to 8.0, 8000 U / g of trypsin was added, and the mixture was placed in a constant-temperature water bath oscillator at 40°C for 2 h. After the enzymolysis was completed, enzyme inactivation treatment was performed at 90°C for 15 min. After cooling to room temperature, the pH value was adjusted to 7.0, and centrifugation was performed to obtain a supernatant. The supernatant was dried to obtain pigeon blood enzymolysis polypeptide. The pigeon blood enzymolysis polypeptide was pigeon blood antioxidant and anti-aging active polypeptide.

[0042] Example 3

[0043] The preparation method of the pigeon blood antioxidant and anti-aging active peptide specifically comprises the following steps:

[0044] (1) The pigeon blood was added with 0.4% sodium citrate solution for anticoagulation, and was subjected to ultrasonic treatment at 20 kHz and 100 W for 3 min by using an ultrasonic cell disrupter to dissolve hemoglobin in the pigeon blood;

[0045] (2) After the pretreatment, a certain amount of deionized water was added, the blood-water ratio was adjusted to 1:2, the pH value was adjusted to 8.0, 8000 U / g of trypsin was added, and the mixture was placed in a constant-temperature water bath oscillator at 40°C for 2 h. After the enzymolysis was completed, enzyme inactivation treatment was performed at 90°C for 15 min. After cooling to room temperature, the pH value was adjusted to 7.0, and centrifugation was performed to obtain a supernatant. The supernatant was dried to obtain pigeon blood enzymolysis polypeptide. The pigeon blood enzymolysis polypeptide was pigeon blood antioxidant and anti-aging active polypeptide.

[0046] Different hemolysis pretreatment methods were adopted in Examples 1-3, and the hemoglobin content released in step (1) of Examples 1-3 was determined to optimize the hemolysis method of blood cells.

[0047] The results of the hemolysis pretreatment are shown in Table 1. Figure 1 As shown in Table 1, repeated freezing and thawing, homogenization, and ultrasonic treatment can all cause the rupture of blood cells and the release of hemoglobin. Among them, the ultrasonic pretreatment has the highest hemoglobin dissolution amount. Figure 1

[0048] Table 1. Protein recovery rate, degree of hydrolysis, and activity results of the pigeon blood active peptides prepared in Examples 1-3

[0049]

[0050] Example 4

[0051] On the basis of Example 3, the pigeon blood protein hydrolysis conditions were optimized to obtain polypeptides with good antioxidant and anti-aging activities. Single-factor and orthogonal experiments were adopted in the optimization experiment, and the effects of the type of protease, blood-water ratio, enzyme dosage, and enzymolysis time on the protein recovery rate, degree of hydrolysis, DPPH and ABTS antioxidant activities, and tyrosinase inhibitory activity of the enzymolysis products were considered.​

[0052] First, 30 mL of pigeon blood (from a 21-day-old white-feathered king pigeon) was added to a 0.4% sodium citrate solution for anticoagulation. After hemolysis pretreatment, a certain amount of deionized water was added, and the pH was adjusted to the optimal pH of the enzyme. Protease was added, and the sample was placed in a constant temperature water bath shaker for constant temperature enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 90-100℃. After cooling to room temperature, the pH was adjusted to 7.0, and the sample was centrifuged (4℃, 10000r / min, 15min). The supernatant was collected for subsequent index determination. After drying the supernatant, the active polypeptide of pigeon blood was obtained.

[0053] 4.1 Single-factor experiment

[0054] Investigation of protease types: Pigeon blood after hemolysis was hydrolyzed at a blood-to-water ratio of 1:2, with an enzyme dosage of 10000 U / g and a hydrolysis time of 3 hours. Alkaline protease (pH 8.5), flavor protease (pH 7.5), neutral protease (pH 7.5), complex protease (pH 7.0), and papain (pH 7.0) were used at 50℃, while trypsin (pH 7.5) and pepsin (pH 2.0) were used at 37℃. The effects of different proteases on the hydrolysis of pigeon blood were investigated.

[0055] Blood-to-water ratio study: Under the conditions of 10000 U / g enzyme dosage and 3h enzymatic hydrolysis time, pepsin was used for enzymatic hydrolysis at pH 2.0 and 37℃ to investigate the effect of different blood-to-water ratios (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5) on the enzymatic hydrolysis effect of pigeon blood.

[0056] Enzyme dosage investigation: Under the conditions of blood-to-water ratio of 1:1 and enzymatic hydrolysis time of 3 hours, pepsin was used for enzymatic hydrolysis at pH 2.0 and 37℃ to investigate the effect of different enzyme dosages (4000, 6000, 8000, 10000, 12000 U / g) on ​​the enzymatic hydrolysis effect of pigeon blood.

[0057] Investigation of enzymatic hydrolysis time: Under the conditions of blood-to-water ratio of 1:1 and enzyme dosage of 12000 U / g, pepsin was used for enzymatic hydrolysis at pH 2.0 and 37℃ to investigate the effect of different enzymatic hydrolysis times (1, 2, 3, 4, 5 h) on the enzymatic hydrolysis effect of pigeon blood.

[0058] 4.2 Orthogonal Experiment

[0059] Further, a three-factor, three-level (L9) orthogonal experiment was conducted to determine the optimal enzymatic hydrolysis process for the factors identified in the single-factor experiments that had a significant impact on the enzymatic hydrolysis effect. The experimental design is shown in Table 2.

[0060] Table 2. Factor Level Design of Orthogonal Experiment for Pigeon Blood Enzymatic Hydrolysis

[0061]

[0062] 4.3 Determination of protein recovery and degree of hydrolysis

[0063] The protein content in the supernatant of pigeon blood enzymolysis was determined by the biuret method. The degree of hydrolysis (DH) was determined by the OPA method.

[0064] Protein recovery (PR, %) = (protein content in the supernatant / total protein content in the pigeon blood enzymolysis solution) x 100%

[0065] 4.4 Antioxidant activity of the supernatant of pigeon blood enzymolysis

[0066] The ABTS and / or DPPH radical scavenging capacity of the supernatant of enzymolysis was evaluated.

[0067] 4.5 Tyrosinase inhibitory activity

[0068] 100 μL of pigeon blood enzymolysis supernatant at a certain concentration was mixed with an equal volume of 125 U / mL tyrosinase (dissolved in 0.05 mol / L PBS, pH 6.8), incubated at 25°C for 5 min, and then an equal volume of 10 mmol / L L-DOPA (dissolved in 0.05 mol / L PBS, pH 6.8) was added, and the resulting reaction mixture was incubated at 25°C for another 5 min. The content of dopachrome in the reaction mixture was determined by detecting the absorbance at 475 nm. The tyrosinase inhibitory activity was calculated according to the following formula:

[0069]

[0070] In the formula, A is the tyrosinase mixture without the sample; B is the mixture without the sample and tyrosinase; C is the mixture of the sample and tyrosinase; and D is the mixture containing the sample but not containing tyrosinase.

[0071] 4.6 Experimental results and discussion

[0072] Enzyme type investigation results: The effects of different enzymes on pigeon blood protein enzymolysis are shown in Figure 2 From the figure, it can be seen that the DPPH and ABTS radical scavenging activity and tyrosinase inhibitory rate of the supernatant after pepsin and trypsin hydrolysis are relatively high, but the protein recovery rate of trypsin is relatively low, so pepsin is the optimal enzyme for pigeon blood enzymolysis.

[0073] Blood-water ratio investigation results: The effects of blood-water ratio on pigeon blood enzymolysis are shown in Figure 3The results of the enzyme addition amount investigation are shown in Table 4. As shown in Table 4, the higher the enzyme addition amount, the better the enzyme hydrolysis effect of the pigeon blood enzyme hydrolysis supernatant, and the protein recovery rate, the degree of hydrolysis, and the ABTS free radical scavenging activity and tyrosinase scavenging activity of the enzyme hydrolysis supernatant are the highest at an enzyme addition amount of 12000 U / g.

[0074] The results of the enzyme addition amount investigation are shown in Table 4. As shown in Table 4, the higher the enzyme addition amount, the better the enzyme hydrolysis effect of the pigeon blood enzyme hydrolysis supernatant, and the protein recovery rate, the degree of hydrolysis, and the ABTS free radical scavenging activity and tyrosinase scavenging activity of the enzyme hydrolysis supernatant are the highest at an enzyme addition amount of 12000 U / g. Figure 4

[0075] The results of the enzyme hydrolysis time investigation are shown in Table 5. As shown in Table 5, the ABTS free radical scavenging activity and tyrosinase inhibitory activity of different enzyme hydrolysis times are: 5h > 1h > 4h > 2h > 3h, and therefore, the enzyme hydrolysis time of 5h is the best time for pigeon blood enzyme hydrolysis. Figure 5

[0076] The results of the orthogonal experiment are shown in Table 3. In the 9th experiment, the blood-water ratio is 1:1.2, the enzyme addition amount is 12000 U / g, and the enzyme hydrolysis time is 4h, and the obtained tyrosinase inhibition rate reaches the maximum value of 45.93%, and at this time, the ABTS clearance rate also reaches a high value of 83.84%, and therefore, this enzyme hydrolysis condition is selected as the best enzyme hydrolysis process of pepsin.

[0077] Table 3 Results and analysis of pigeon blood enzyme hydrolysate orthogonal optimization experiment

[0078]

[0079] Based on the above results, the best treatment condition for preparing antioxidant and anti-aging active peptides from pigeon blood enzyme hydrolysis is: pigeon blood 20kHz 100W ultrasonic treatment for 5min, blood-water ratio 1:1.2, and pepsin 37℃, pH 2.0, enzyme addition amount 12000 U / g, and enzyme hydrolysis time 4h.

[0080] The enzyme hydrolysis supernatant prepared under the optimal condition was analyzed for amino acid and molecular weight distribution, and it was found that the pigeon blood active peptides obtained under this process were mainly composed of peptide segments with a molecular weight of less than 5000kDa (accounting for 78.98%), and had a high content of essential amino acids and antioxidant and anti-aging active amino acids, reaching 42.01% and 27.22%, respectively.

[0081] Example 5, verification of the antioxidant and anti-aging activity of pigeon blood active peptides on D-galactose-induced aging mice

[0082] 5.1 Modeling and intervention

[0083] ​​SPF Kunming male mice were selected for the experiment, and were divided into 6 groups: normal group, model group, positive control group (piracetam, 400 mg / Kg.bw / d), pigeon blood active peptide group (obtained under the best treatment condition of Example 4, 300 mg / Kg.bw / d), pigeon blood active peptide group (obtained under the best treatment condition of Example 4, 600 mg / Kg.bw / d) and pigeon blood active peptide group (obtained under the best treatment condition of Example 4, 1200 mg / Kg.bw / d), 10 mice in each group. Except for the normal group injected with normal saline, the mice in the other groups were given D-galactose by subcutaneous injection for 4 weeks, and the drug volume was 10 mL / kg body weight; the pigeon blood active peptide group and the positive control group were simultaneously fed with the corresponding test drugs, and the feeding volume was 10 mL / kg, and the normal group and the model group were fed with the same volume of water. During the experiment, all mice were fed with standard feed, and the water was changed every other day, and the mouse cage was cleaned 4 times / week.

[0084] 5.2 Index determination

[0085] At 1 h after the last administration, the mice were intraperitoneally anesthetized with 3% sodium pentobarbital, and orbital venous blood was taken, and the serum was collected, and after cold storage for 2-3 hours, the serum was separated by centrifugation at 3000 r / min for 10 min, and the serum was separated and frozen in liquid nitrogen, and stored at -80℃ to avoid repeated freezing and thawing. After the mice were bled, they were executed by cervical dislocation, decapitated, and the skull was stripped, and the whole brain tissue was taken out under an ice tray, and the excess blood was absorbed with filter paper, and the brain tissue was quickly frozen in liquid nitrogen and stored at -80℃ for later use. Before the experiment, the tissue samples were thawed, washed with pre-cooled normal saline, and homogenized in an ice bath. The brain homogenate was centrifuged at 3000 rpm, 4℃ for 15 min, and the supernatant was obtained, i.e. 10% brain homogenate.

[0086] The contents of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-px) and NO in the serum and brain homogenate were determined by using the kit. The levels of monoamine oxidase (MAO), advanced glycation end products (AGEs) and Klotho in the brain homogenate were also determined.

[0087] 5.3 Results and discussion

[0088] The effect of pigeon blood active peptide on blood antioxidant indexes of aging mice was as follows: Figure 6The results are shown in Figure 2. As can be seen from the figure, compared with the normal group (Control), the activities of SOD and GSH-Px in the serum of the model group (Model) mice were significantly reduced (P<0.05), while the contents of MDA and NO were significantly increased (P<0.05). MDA is one of the markers indicating the severity of lipid peroxidation, indicating that the cells of the body are severely attacked by free radicals. After the administration of different doses of pigeon blood active peptide and positive drugs to the aging mice, compared with the Model group, the activities of SOD and GSH-Px were significantly improved, and the contents of NO and MDA were significantly reduced (P<0.05), and showed a dose-dependent relationship. In particular, the SOD and GSH-Px activities of the mice in the middle dose pigeon blood active peptide group were the highest, almost reaching the levels of the Control group and the piracetam group (positive drug). Different doses of pigeon blood active peptide can effectively enhance the activities of SOD and GSH-Px in the serum of mice and reduce the contents of MDA and NO, indicating that a certain dose of pigeon blood active peptide can better improve the oxidative stress level in the body of D-galactose-induced aging mice, thereby delaying the tissue or cell damage caused by free radicals.

[0089] Brain aging is closely related to oxidative stress. As the most active organ of the human body, it highly depends on oxygen to produce energy. Among various theories describing the mechanism of brain aging, the free radical theory is the most widely accepted description of this process. The effects of pigeon blood active peptide on the biochemical indicators of brain tissue of aging mice are shown in Figure 3. Figure 7 As shown in the figure, after D-galactose modeling, the activities of SOD and GSH-Px in the brain tissue of mice were significantly decreased (P<0.05), and the contents of MDA and NO were significantly increased (P<0.05), indicating that the modeling was successful. After the mice were injected with D-galactose in the neck, compared with the Model group, the activities of SOD and GSH-Px in the brain tissue of mice in the middle dose group and the high dose group of pigeon blood active peptide were significantly improved, and the contents of MDA and NO were significantly decreased (P<0.05). The indicators of the low dose group had a certain degree of improvement compared with the Model group, but there was no significant difference (P>0.05), indicating that different doses of pigeon blood active peptide intervention can effectively increase the activities of SOD and GSH-Px in the brain tissue, exert its strong free radical scavenging capacity, reduce the deposition of MDA and NO in the brain tissue, and improve the oxidative stress level of the brain tissue of aging mice.

[0090] Monoamine oxidase (MAO) is a key intracellular enzyme present in the outer membrane of mitochondria and the central nervous system, responsible for the decomposition of neuroactive amines and vasoactive amines, and can promote the aging of the nervous system, thereby leading to the degeneration of brain function (Mathimaran et al., 2021). From Figure 7 As can be seen from Figure 4 (E), the MAO content of the pigeon blood active peptide group was significantly lower than that of the Model group (P<0.05), and showed a certain dose-dependent relationship.

[0091] Klotho is a single-channel transmembrane protein, and its deficiency will shorten the life span of mice and exhibit a variety of phenotypes similar to human premature aging, such as motor neuron and hippocampal degeneration, cognitive impairment, hair loss, etc. Regulating Klotho expression is an important strategy for anti-aging research. In mouse and human studies, Klotho levels decrease with age and are associated with shorter lifespan. From Figure 7 As can be seen in (G), the Klotho level in the brain tissue of mice injected with D-gal for a long time will be significantly reduced (P<0.05), and after gavage with different doses of pigeon blood active peptide, the Klotho protein level in the brain tissue of mice can be effectively improved, and a certain dose effect is shown.

[0092] AGEs are a group of heterogeneous reaction products formed between the amino groups of proteins and the aldehyde groups derived from carbohydrates through non-enzymatic glycosylation of reducing sugars such as D-glucose and d-galactose. AGEs can accelerate the aging process and participate in the early stages of age-related diseases, including neurodegenerative diseases, kidney failure, arthritis, and age-related macular degeneration, etc. From Figure 7 As can be seen in (F), compared with the Control group, the AGEs content of the Model group is significantly increased (P<0.05), and the AGEs content in the brain tissue of mice in the medium and high dose pigeon blood active peptide groups is significantly lower than that of the Model group (P<0.05), indicating that pigeon blood active peptide can effectively block the increase of AGEs in D-gal mice.

[0093] The above results show that pigeon blood active peptide can significantly improve the antioxidant level of D-gal-induced aging mice, improve the oxidative stress state of aging mice, and regulate the expression of MAO, AGEs, Klotho and other aging-related factors or proteins in brain tissue, thereby effectively delaying aging and playing an anti-aging effect.

[0094] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for preparing pigeon blood active peptide, characterized in that It comprises the following steps: (1) Pigeon blood is added with an anticoagulant and subjected to hemolysis pretreatment to dissolve pigeon hemoglobin; (2) After the pretreatment, deionized water is added to adjust the blood-water ratio and pH value, a protease is added for enzymolysis, and after the enzymolysis is completed, the enzyme is inactivated, and after being cooled to room temperature, the pH value is adjusted to 7.0, centrifugation is performed to obtain supernatant, and after drying, pigeon blood enzymolysis polypeptide is obtained; The protease is pepsin or trypsin; The pepsin enzymolysis conditions are: temperature is 35-40 °C, pH value is 1.5-2.0, enzyme addition amount is 4000-12000 U / g, and enzymolysis time is 1-5 h; The trypsin enzymolysis conditions are: temperature is 35-40 °C, pH value is 7.0-8.0, enzyme addition amount is 4000-12000 U / g, and enzymolysis time is 1-5 h.

2. The method of claim 1, wherein: In step (2), the blood-water ratio is the mass ratio of pigeon blood to deionized water of 1:0.5-1:2.

5.

3. The method of claim 1, wherein: The hemolysis pretreatment conditions are: repeated freezing and thawing at-15--20 °C for 1-3 times, or homogenization by a high-speed shearing machine at 5000-8000 rpm for 3-5 min, or ultrasonic treatment by an ultrasonic cell disrupter at 20 kHz and 100-300 W for 3-5 min.

4. The method of claim 1, wherein: In step (1), the anticoagulant is a 0.4% sodium citrate solution.

5. The method of claim 1, wherein: The enzyme inactivation conditions are: temperature is 90-100 °C, and time is 10-15 min.

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