A preparation method, sequence composition and application of an antioxidant short peptide from yak hide

Abstract

The present invention discloses a preparation method, sequence composition and application of antioxidant short peptides from yak skin, belonging to the field of bioactive peptide processing. A preparation method, sequence composition and application of antioxidant short peptides from yak skin include yak skin short peptides with a molecular weight of less than 1 kDa, comprising 27 peptide sequences and having antioxidant activity. The preparation process is as follows: First, 1 g of yak skin collagen powder is dissolved in 180 ml of ultrapure water and extracted in a water bath at 75 °C for 5 hours. The supernatant is obtained by centrifugation to obtain a yak skin polypeptide solution. The above solution is ultrafiltered through a 1KD ultrafiltration membrane, and the filtrate is collected and freeze-dried. Then, gel separation and purification are used. It can be verified through in vitro and in vivo experiments that the yak skin short peptides prepared by this method can not only reduce myocardial injury, but also inhibit oxidative stress reactions, have a certain protective effect on the injury caused by myocardial ischemia-reperfusion, and also play a greater role in enhancing antioxidant activity and reducing oxidative stress injury.

Description

Technical Field

[0001] The present invention relates to the field of bioactive peptide processing, and more specifically, to a preparation method, sequence composition and application of antioxidant short peptides from yak skin. Background Art

[0002] Currently, there is an increasing interest in functional foods that promote health, dietary supplements and pharmaceutical preparations containing bioactive peptides extracted from food proteins. In addition to the most classic antioxidant activity, polypeptides also have the functions of regulating immunity and biological activities such as anti-tumor, reducing blood pressure and blood lipids, and anti-bacteria, which can adjust and control various functions of the human body. Bioactive peptides usually consist of 2 to 15 amino acids. Compared with proteins, active peptides have unique advantages, such as good solubility, high bioavailability, low gelation property, and low viscosity, and have good processing performance. Related products developed with polypeptides as prototypes have been widely used in various fields such as medicine and health, food and health care, beauty and cosmetics, and biological materials. Some related studies have verified in clinical trials that the use of some bioactive peptides in foods, nutritional products and drugs has a protective and therapeutic effect on promoting health and relieving diseases. Therefore, appropriate supplementation of specific active peptides not only meets the human body's need for essential amino acids from a nutritional perspective, but also has a profound impact on enhancing physical fitness, improving the condition, and resisting aging.

[0003] The yak is one of the precious cattle breeds unique to the high-altitude regions of China. Through the selection of nature, it has the ability to adapt to the plateau climate with low temperature and low oxygen. The yak population shows relatively good adaptability to the harsh living conditions on the plateau that most livestock cannot adapt to, and has obtained basic hypoxic adaptability in terms of physiological and biochemical indexes, gene functions and anatomical structures. Thus, many parts of the whole body have the value of in-depth development. Yak skin collagen peptide is a peptide mixture obtained from yak skin through processes such as preparation, separation and purification. Currently, the research and development of Chinese and Western medicines for plateau hypoxia have achieved fruitful results. However, Western medicines have more or less side effects, and Chinese herbal medicines are relatively expensive. In addition to drugs, polypeptide products not only play a role in preventing or treating diseases, but also have fewer side effects and wide-ranging effects. Therefore, the development of animal-derived bioactive peptides has gradually emerged, and the antioxidant research and development and utilization of bioactive peptides have become the focus. Summary of the Invention

[0004] 1. Technical Problems to be Solved

[0005] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a preparation method, sequence composition and application of antioxidant short peptides from yak skin, which can prove through in vitro and in vivo experiments that the short peptides from yak skin prepared by this method can not only reduce myocardial injury, but also inhibit oxidative stress response, have a certain protective effect on the injury caused by myocardial ischemia-reperfusion, and also play a greater role in enhancing antioxidant activity and reducing oxidative stress injury.

[0006] 2. Technical solution

[0007] To solve the above problems, the present invention adopts the following technical solutions.

[0008] A preparation method of antioxidant short peptides from yak skin, including short peptides from yak skin, the molecular weight of the short peptides from yak skin is less than 1KDa, contains 27 peptide sequences, and has antioxidant activity.

[0009] Further, the preparation method of the antioxidant short peptides from yak skin includes the following steps:

[0010] S1. Short peptide preparation: Take 1g of yak skin collagen powder and add it to 180ml of pure water, dissolve it in a water bath at 75°C, stir it evenly for extraction for 5h, filter it by suction, and take its dissolved solution; filter it with a 1KD ultrafiltration membrane, collect the polypeptide solution with a molecular weight less than 1KDa, and freeze-dry it under vacuum at -40°C to obtain a polypeptide freeze-dried powder.

[0011] S2. Column chromatography: After fully swelling the SephadexG-15 packing material, uniformly pack it into a glass chromatography column (specification: 1.6cm×150cm), prepare the yak skin peptide powder obtained in step S1 into a 2mg / mL solution with ultrapure water as the solvent, the sample loading volume is 4mL, elute it with ultrapure water at a flow rate of 0.5mL / min, and at the same time detect it with a UV detector at 280nm. A total of 4 elution peaks are detected, collect the elution peaks and freeze-dry them, measure the antioxidant activity of each component, take the component with the highest activity, and store it at low temperature for later use;

[0012] S3. Sequence identification: After separating the sample obtained in step S2 by capillary high performance liquid chromatography, perform mass spectrometry analysis with a QExactive mass spectrometer. Analysis duration: 60min. Detection method: positive ion; the mass-to-charge ratio of polypeptides and polypeptide fragments is collected according to the following method: 10 fragment spectra are collected after each full scan.

[0013] Further, the sequence composition of the antioxidant short peptides from yak skin includes that the short peptides from yak skin have a certain scavenging effect on 1,1-diphenyl-2-trinitrophenylhydrazine, hydroxyl radicals and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0014] Furthermore, the yak hide antioxidant short peptide is used in a medicament for protecting against myocardial ischemia-reperfusion injury.

[0015] 3. Beneficial effects

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] This solution can be verified through in vivo and in vitro experiments that the yak hide short peptide prepared by this method can not only reduce myocardial injury, but also inhibit the oxidative stress response, and has a certain protective effect on the injury caused by myocardial ischemia-reperfusion, and also plays a greater role in enhancing antioxidant activity and reducing oxidative stress injury. Description of the drawings

[0018] Figure 1 It is a schematic diagram of the separation effect of the yak hide short peptide of the present invention after separation by a gel column;

[0019] Figure 2 It is a schematic diagram of the test results of the antioxidant activity of the yak hide short peptide of the present invention;

[0020] Figure 3 It is a schematic diagram of the total ion current of the LC-MSMS mass spectrometry identification of the yak hide short peptide of the present invention;

[0021] Figure 4 It is a schematic diagram of the influence of the yak hide short peptide of the present invention on the myocardial infarction area of myocardial ischemia-reperfusion;

[0022] Figure 5 It is a sequence list of 27 peptide segments of the yak hide short peptide of the present invention. Detailed implementation manners

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention; obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Example 1:

[0025] Please refer to Figures 1-4 , a method for preparing, sequence composition and application of a yak hide antioxidant short peptide, including a yak hide short peptide, the molecular weight of the yak hide short peptide is less than 1 KDa, it contains 27 peptide segment sequences, and has antioxidant activity.

[0026] The method for preparing the yak hide antioxidant short peptide includes the following steps:

[0027] S1. Short peptide preparation: Take 1 g of human yak skin collagen powder and add it to 180 ml of pure water. Dissolve it in a water bath at 75°C, stir it evenly for extraction for 5 h, filter by suction, and take the dissolved solution. Filter it with a 1 KD ultrafiltration membrane, collect the polypeptide solution with a molecular weight less than 1 KDa, and freeze-dry it under vacuum at -40°C to obtain the freeze-dried polypeptide powder.

[0028] S2. Column chromatography: After fully swelling the Sephadex G-15 packing material, uniformly pack it into a glass chromatography column (specification: 1.6 cm × 150 cm). Prepare the yak skin peptide powder obtained in step S1 into a 2 mg / mL solution with ultrapure water as the solvent, with a sample loading volume of 4 mL, elute it with ultrapure water at a flow rate of 0.5 mL / min, and at the same time detect it with a UV detector at 280 nm. A total of 4 elution peaks are detected. Collect the elution peaks and freeze-dry them, measure the antioxidant activity of each component, take the component with the highest activity, and store it at low temperature for standby.

[0029] S3. Sequence identification: After separating the sample obtained in step S2 by capillary high-performance liquid chromatography, perform mass spectrometry analysis with a QExactive mass spectrometer. Analysis duration: 60 min. Detection method: positive ion; The mass-to-charge ratio of polypeptides and polypeptide fragments is collected according to the following method: 10 fragment spectra are collected after each full scan.

[0030] The sequence composition of the antioxidant short peptide from yak skin, including the antioxidant short peptide from yak skin, has a certain scavenging effect on 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radicals, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

[0031] The application of the antioxidant short peptide from yak skin in drugs for protecting against myocardial ischemia-reperfusion injury.

[0032] Preliminary preparation: Weigh yak skin collagen powder with the same mass, change the conditions to set different solvent volumes, extraction temperatures, and extraction times to obtain extraction solutions. After multiple preliminary experiments, it is found that the solvent volume is between 140 - 220 ml; the extraction temperature is between 60 - 80°C; and the enzymatic hydrolysis time is between 2 - 6 h, and the extraction rate is relatively high.

[0033] Example 2:

[0034] Take five portions of 1 g of yak skin collagen powder and stir and extract it in a water bath at 70°C in different volumes (140 ml, 160 ml, 180 ml, 200 ml, 220 ml) of ultrapure water for 4 h, filter by suction, dry the undissolved sample powder, weigh it, calculate the dissolved grams / total weight, and calculate the extraction rate.

[0035] Example 3:

[0036] Take five portions of 1 g of yak hide collagen powder and dissolve it in 180 ml of ultrapure water. Stir and extract it in a water bath at different temperatures (60 °C, 65 °C, 70 °C, 75 °C, 80 °C) for 4 h, then filter by suction. Dry the undissolved sample powder, weigh it, and calculate the dissolved grams / total weight, which is the extraction rate.

[0037] Example 4:

[0038] Take five portions of 1 g of yak hide collagen powder and dissolve it in 180 ml of ultrapure water at 70 °C. Stir and extract it in a water bath for different times (2 h, 3 h, 4 h, 5 h, 6 h), then filter by suction. Dry the undissolved sample powder, weigh it, and calculate the dissolved grams / total weight to calculate the extraction rate.

[0039] Examples 2 - 4 studied the effects of three single factors, namely the solvent volume, extraction time, and extraction temperature, on the extraction rate of yak hide short peptides. The results of Example 2 showed that when the water bath temperature (70 °C) and water bath time (4 h) of the yak hide collagen powder were the same, the dissolved weight was the largest and the extraction rate was the highest at 13.87% when the solvent volume was 180 ml; the results of Example 3 showed that when the solvent volume (180 ml) and water bath time (4 h) of the yak hide collagen powder were the same, the dissolved weight of artificial tiger bone was the largest and the extraction rate was the highest at 11.73% when the water bath temperature was 75 °C; the results of Example 4 showed that when the water bath temperature (70 °C) and solvent volume (180 ml) of the yak hide collagen powder were the same, the dissolved weight was the largest and the extraction rate was the highest at 11.75% when the water bath time was 5 h. In summary, the extraction rate of yak hide collagen powder is the highest at 75 °C, in 180 ml of ultrapure water, and with a water bath time of 5 h.

[0040] Example 5:

[0041] Pass the yak hide peptide solution through ultrafiltration membranes with a molecular weight cut-off of 10 KDa, 5 KDa, 3 KDa, and 1 KDa to remove large molecular fragments. Concentrate the filtrate less than 1 KDa and freeze-dry it to obtain a yak hide short peptide sample.

[0042] After fully swelling the Sephadex G-15 packing material, evenly pack it into a glass chromatography column (with a specification of 1.6 cm × 150 cm). Prepare the yak hide peptide powder obtained in step S1 into a 2 mg / mL solution with ultrapure water as the solvent, with a sample loading volume of 4 mL. Elute it with ultrapure water at a flow rate of 0.5 mL / min, and at the same time detect it with a UV detector at 280 nm. A total of 4 elution peaks are detected. Collect the elution peaks, freeze-dry them, measure the antioxidant activity of each component, take the component with the highest activity, and store it at low temperature for standby.

[0043] The above samples were separated by capillary high performance liquid chromatography and then analyzed by Q Exactive mass spectrometer. Analysis duration: 60 min. Detection mode: positive ion. The mass-to-charge ratios of polypeptides and polypeptide fragments were collected according to the following method: 10 fragment spectra were collected after each full scan.

[0044] Example 6:

[0045] 1) DPPH free radical scavenging experiment:

[0046] Dissolve a certain amount of yak hide short peptides. Take 1 mL of the sample solution and add 4 mL of DPPH solution with a concentration of 0.1 mmol / L. Mix well, and measure its absorbance value after 30 min in the dark at a wavelength of 595 nm. Each group was measured three times.

[0047] 2) ABTS free radical scavenging experiment:

[0048] Prepare an ABTS free radical stock solution and let it stand for about 13 h at room temperature in the dark for later use. Take 0.2 mL of yak hide short peptide solutions with different mass concentrations and 3.8 mL of ABTS solution, mix evenly, let it stand in the dark at room temperature for 6 min, and measure its absorbance at 734 nm. Each group was measured three times.

[0049] 3) ·OH free radical scavenging experiment:

[0050] Prepare Fe2SO4 with a concentration of 9 mmol / L, salicylic acid ethanol solution with a concentration of 9 mmol / L, and H2O2 with a concentration of 8.8 mmol / L. Measure 1 mL of yak hide short peptide solutions with different mass concentrations, add 1 mL of each of the above Fe2SO4 and salicylic acid-ethanol solutions respectively, and finally add 1 mL of H2O2, mix well, react at 37 °C for 1 h, and measure the absorbance at 510 nm. Each group was measured three times.

[0051] Example 7:

[0052] The protective effect of yak hide short peptides on myocardial ischemia-reperfusion injury in mice;

[0053] Thirty KM mice were adaptively fed for one week. The indoor temperature was maintained at 20°C ± 2°C, the humidity of the feeding environment was 50%, and the light was changed according to the natural day-night rhythm. They were randomly divided into 5 groups, with 6 mice in each group: namely, the sham operation group: threading but not ligating, and intraperitoneally injecting normal saline (5 ml / kg) 25 minutes after threading; the MI / R model group: ligating the left anterior descending branch, and intraperitoneally injecting normal saline (5 ml / kg) 5 minutes before reperfusion; the low-dose yak skin short peptide group: ligating the left anterior descending branch, and intraperitoneally injecting (0.5 mg / kg, 5 ml / kg) 5 minutes before reperfusion; the medium-dose yak skin short peptide group: ligating the left anterior descending branch, and intraperitoneally injecting (1 mg / kg, 5 ml / kg) 5 minutes before reperfusion; the high-dose yak skin short peptide group: performing ischemia-reperfusion treatment, and intraperitoneally injecting (2 mg / kg, 5 ml / kg) 5 minutes before reperfusion.

[0054] In this experiment, a mouse MI / R model was established by ligating and then releasing the left anterior descending branch of the coronary artery. After 30 minutes of ischemia and 120 minutes of reperfusion in the mice, the myocardial infarction area was detected by the triphenyltetrazolium chloride staining method, the activity of lactate dehydrogenase in the serum was measured, and the contents of malondialdehyde and superoxide dismutase in the myocardial tissue were determined. The myocardial injury and oxidative stress response were detected by means of the above indexes.

[0055] (1) Effect of yak skin short peptide on myocardial infarction area

[0056] Table 1 Effect of yak skin short peptide on myocardial infarction area

[0057]

[0058] Note: △△△ P < 0.001 vs sham operation group; * P < 0.05, *** P < 0.001 vs model group

[0059] As can be seen from Table 1, compared with the model group, the yak skin short peptide mixture provided by this application can reduce the myocardial infarction area to a certain extent and has a protective effect on the injury caused by myocardial ischemia-reperfusion in mice.

[0060] (2) Yak skin short peptide relieves MI / R-induced myocardial injury and inhibits oxidative stress in the MI / R heart

[0061] Table 2 Biochemical indexes of mice in each group

[0062]

[0063] Note: △△△ P < 0.001 vs sham operation group; * P < 0.05, ** P < 0.01,*** P < 0.001 vs. model group

[0064] As can be seen from Table 2, the study shows that compared with the model group, the values of MDA and LDH in the sham operation group are lower, and the value of SOD is higher, with statistical differences (P < 0.05); compared with the model group, the values of MDA and LDH in the yak skin short peptide group are significantly decreased, with statistical differences (P < 0.05), and the value of SOD also shows an upward trend, indicating that the yak skin short peptide mixture provided in the embodiments of the present application can not only reduce myocardial injury, but also inhibit oxidative stress response, and has a certain protective effect on the injury caused by myocardial ischemia-reperfusion.

[0065] The above is only the preferred specific implementation manner of the present invention; however, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its improved concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. Use of yak skin antioxidant short peptide in the preparation of a drug for improving myocardial ischemia-reperfusion injury, Characterized in that: The yak skin antioxidant short peptide comprises a short peptide with an amino acid sequence as shown in SEQ ID NO.1-27.

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