Yak skin oligopeptide, preparation method and application thereof, and functional food or medicine
The yak hide oligopeptides RDIFL, FRVP, RDIFL, and FDLRF, prepared by solid-phase synthesis, solve the problems of expensive and side-effect-prone existing α-glucosidase inhibitors, achieve effective inhibition of α-glucosidase, and provide a safe functional food and pharmaceutical solution.
Patent Information
- Application Number
- CN202511316581.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-16
- Publication Date
- 2025-12-05
- Estimated Expiration
- 2045-09-16
AI Technical Summary
Existing alpha-glucosidase inhibitors, such as acarbose, are expensive and have side effects. The search for natural and effective alpha-glucosidase inhibitors to delay postprandial hyperglycemia symptoms is an important area of research in type 2 diabetes.
Yak hide oligopeptides RDIFL, FRVP, RDIFL, and FDLRF were prepared using a solid-phase synthesis method. Their binding ability to α-glucosidase was verified by molecular docking technology, and they were applied to functional foods and pharmaceuticals.
Yak hide oligopeptides exhibit excellent inhibitory activity against α-glucosidase. Their small molecular weight allows for direct absorption by the intestines, effectively delaying carbohydrate digestion and absorption, reducing postprandial blood glucose, broadening the diversified applications of yak hide, and providing safe functional food and pharmaceutical solutions.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of bioactive peptides, in particular to a yak skin oligopeptide as well as a preparation method and application thereof, and a functional food or drug. BACKGROUND
[0002] Type 2 diabetes mellitus (T2DM) is a disease characterized by persistent hyperglycemia and affected by the synergistic effect of genetic and environmental factors, which is proved to be one of the most common and fastest growing diseases in the world. Its characteristics are insulin deficiency due to dysfunction of pancreatic beta cells and insulin resistance. The main symptoms include elevated blood glucose and insulin resistance.
[0003] Alpha-glucosidase is an important enzyme in the body, mainly distributed in the brush border of small intestinal cells. After eating, the carbohydrates in the food are first hydrolyzed into shorter polysaccharides under the catalysis of amylase in saliva, and then part of the hydrolyzed starch is further converted by amylase secreted by the pancreas. When the food reaches the small intestine, alpha-glucosidase is responsible for hydrolyzing the alpha-1,4 glycosidic bond of oligosaccharides and disaccharides in the food, so as to convert them into glucose for absorption by the human body. Therefore, by using alpha-glucosidase inhibitors to limit the catalytic activity of alpha-glucosidase, the digestion process of carbohydrates in the diet can be effectively delayed, thereby regulating the postprandial hyperglycemia symptoms of T2DM patients. However, the clinically used alpha-glucosidase inhibitors such as acarbose and voglibose are expensive and have side effects such as abdominal pain and diarrhea. Therefore, finding natural substances with alpha-glucosidase inhibitory activity has become an important field of interest in diabetes research. SUMMARY
[0004] The present application aims at the technical defects in the prior art, and provides a yak skin oligopeptide.
[0005] Another object of the present application is to provide a preparation method of the yak skin oligopeptide.
[0006] Another object of the present application is to provide an application of the yak skin oligopeptide.
[0007] Another object of the present application is to provide a functional food.
[0008] Another object of the present application is to provide a drug.
[0009] The technical scheme adopted by the present application to achieve the objects of the present application is as follows:
[0010] A yak skin oligopeptide, the yak skin oligopeptide comprising at least one of RDIFL, FRVP, RDILF and FDLRF, wherein the amino acid sequence of the RDIFL is arginine-aspartic acid-isoleucine-phenylalanine-leucine; the amino acid sequence of the FRVP is phenylalanine-arginine-valine-proline; the amino acid sequence of the RDILF is arginine-aspartic acid-isoleucine-leucine-phenylalanine; and the amino acid sequence of the FDLRF is phenylalanine-aspartic acid-leucine-arginine-phenylalanine.
[0011] In the above technical solution, the yak skin oligopeptide is a bioactive peptide taking yak skin as raw material.
[0012] In the above technical solution, the RDIFL, FRVP, RDILF and FDLRF are all synthesized by solid phase synthesis method.
[0013] Another aspect of the present application further comprises a preparation method of the yak skin oligopeptide, and the preparation method is solid phase synthesis method.
[0014] Another aspect of the present application further comprises a preparation method of the yak skin oligopeptide, and the preparation method comprises the following steps:
[0015] Step 1, dissolving yak skin collagen peptide in water, then adding ethanol to obtain a mixture, standing and precipitating, centrifuging to obtain supernatant, concentrating, drying to obtain yak skin crude peptide;
[0016] Step 2, dissolving the yak skin crude peptide obtained in step 1 in ultrapure water, separating and purifying by gel chromatography, taking aqueous solution as eluent, collecting effluent, concentrating, drying to obtain yak skin oligopeptide.
[0017] In the above technical solution, in step 1, the volume concentration of the ethanol in the mixture is 75% to 85%.
[0018] In the above technical solution, in step 1, the standing and precipitating time is 60 to 72 hours.
[0019] In the above technical solution, in step 1, the centrifugation speed is 6000 to 8000 r / min, and the centrifugation time is 15 to 20 minutes.
[0020] In the above technical solution, in step 1 and step 2, the drying is freeze-drying.
[0021] In the above technical solution, in step 2, the chromatographic column of the gel chromatography is TK-Col TA-GF30, and the detection wavelength of the ultraviolet detector is 220 nm.
[0022] In the above technical solution, in step 2, when separation and purification are performed by gel chromatography, the effluent collected at 45-140 min is concentrated.
[0023] Another aspect of the present application also includes the application of the yak skin oligopeptide as an inhibitory peptide of alpha-glucosidase, and the binding energy of the yak skin oligopeptide and alpha-glucosidase is verified by molecular docking technology, and the binding energy is less than-8.9 kcal / mol.
[0024] Another aspect of the present application also includes the application of the yak skin oligopeptide in the preparation of a drug for preventing and treating type 2 diabetes.
[0025] Another aspect of the present application also includes a functional food comprising the yak skin oligopeptide.
[0026] Another aspect of the present application also includes a drug comprising the yak skin oligopeptide.
[0027] Compared with the prior art, the present application has the following beneficial effects:
[0028] 1. The RDIFL, FRVP, RDILF and FDLRF four yak skin oligopeptides of the present application all belong to oligopeptides, and compared with other hypoglycemic peptides, the molecular weight is smaller, and the oligopeptides can be directly absorbed by the intestinal tract to delay the increase of blood glucose;
[0029] 2. The oligopeptides RDIFL, FRVP, RDILF and FDLRF of the present application have excellent inhibitory activity on alpha-glucosidase, and the half-inhibitory concentrations are 10.107 mmol / L, 13.063 mmol / L, 8.653 mmol / L and 12.369 mmol / L respectively, which can effectively delay the digestion and absorption of carbohydrates and reduce postprandial blood glucose;
[0030] 3. The present application clarifies the binding mechanism of the yak skin oligopeptide and alpha-glucosidase by molecular docking technology, which is of great significance for the application of the yak skin oligopeptide in improving type 2 diabetes drugs;
[0031] 4. The present application widens the diversified application of yak skin, fully releases its economic and ecological value, and has important significance for safer functional food for the auxiliary treatment of type 2 diabetes. BRIEF DESCRIPTION OF DRAWINGS
[0032] Figure 1 is the gel chromatogram of the yak skin oligopeptide in examples 2-4 of the present application.
[0033] Figure 2 is the alpha-glucosidase inhibition rate of the yak skin oligopeptide in examples 2-4 of the present application.
[0034] Figure 3 is a high performance liquid chromatogram of the oligopeptide RDIFL of the present application.
[0035] Figure 4 is a mass spectrum of the oligopeptide RDIFL of the present application.
[0036] Figure 5 is a high performance liquid chromatogram of the oligopeptide FRVP of the present application.
[0037] Figure 6 is a mass spectrum of the oligopeptide FRVP of the present application.
[0038] Figure 7 is a high performance liquid chromatogram of the oligopeptide RDILF of the present application.
[0039] Figure 8 is a mass spectrum of the oligopeptide RDILF of the present application.
[0040] Figure 9 is a high performance liquid chromatogram of the oligopeptide FDLRF of the present application.
[0041] Figure 10 is a mass spectrum of the oligopeptide FDLRF of the present application.
[0042] Figure 11 is a global image of molecular docking of the oligopeptide RDIFL of the present application with α-glucosidase and a molecular interaction diagram.
[0043] Figure 12 is a global image of molecular docking of the oligopeptide FRVP of the present application with α-glucosidase and a molecular interaction diagram.
[0044] Figure 13 is a global image of molecular docking of the oligopeptide RDILF of the present application with α-glucosidase and a molecular interaction diagram.
[0045] Figure 14 is a global image of molecular docking of the oligopeptide FDLRF of the present application with α-glucosidase and a molecular interaction diagram.
[0046] Figure 15 is a graph showing the change in fasting blood glucose level of mice in each group in the continuous administration for 30 days in Application Example 4.
[0047] Figure 16 is a graph showing the change in blood glucose level of mice in each group at 0, 0.5, 1, 1.5, 2 h after oral administration of glucose in Application Example 4.
[0048] Figure 17 is the area under the curve of oral glucose tolerance of mice in each group in Application Example 4.
[0049] The α-glucosidase used in the following specific embodiments was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., DMSO (dimethyl sulfoxide) was purchased from Shanghai Haohong Biomedical Technology Co., Ltd., and the four oligopeptides RDIFL, FRVP, RDILF and FDLRF were synthesized by Hefei Senr Biotechnology Co., Ltd. through solid-phase synthesis. DETAILED DESCRIPTION
[0050] The application will be further described below in conjunction with specific examples. It should be understood that the specific examples described herein are merely used to explain the application and are not intended to limit the application.
[0051] Example 1
[0052] A yak skin oligopeptide, the yak skin oligopeptide comprising at least one of four oligopeptides whose amino acid sequences are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
[0053] SEQ ID NO. 1:
[0054] RDIFL.
[0055] SEQ ID NO. 2:
[0056] FRVP.
[0057] SEQ ID NO. 3:
[0058] RDILF.
[0059] SEQ ID NO. 4:
[0060] FDLRF.
[0061] The molecular weight of RDIFL is 662.79 Da; the molecular weight of FRVP is 517.63 Da; the molecular weight of RDILF is 662.79 Da; and the molecular weight of FDLRF is 696.80 Da.
[0062] The four peptides of RDIFL, FRVP, RDILF and FDLRF all belong to oligopeptides, have small molecular weight, can be directly absorbed by the intestinal tract, specifically bind to a-glucosidase, and play a role in delaying the increase of blood glucose. Among them, the docking binding energy of RDIFL and a-glucosidase is-9.8 kcal / mol; the docking binding energy of FRVP and a-glucosidase is-9.4 kcal / mol; the docking binding energy of RDILF and a-glucosidase is-9.4 kcal / mol; the docking binding energy of FDLRF and a-glucosidase is-9.3 kcal / mol, therefore, the four oligopeptides all have good inhibitory effect on a-glucosidase, and can prevent and treat type 2 diabetes.
[0063] Example 2
[0064] As shown in the following, Figures 1-2 The present embodiment provides a preparation method of yak skin oligopeptide, comprising the following steps:
[0065] Step 1, sugar removal: after dissolving the yak skin collagen peptide in water, 4 times volume of anhydrous ethanol is added to obtain a mixture, which is allowed to stand for precipitation for 72 h, centrifuged at 6000 r / min for 15 min, and the supernatant is taken, concentrated by rotary evaporation at 55 r / min at 50℃, and freeze-dried to obtain yak skin crude peptide (powder);
[0066] Step 2, the yak skin crude peptide obtained in step 1 is dissolved in ultrapure water, separated and purified by TK-Col TA-GF30 gel chromatography, the loading concentration is 10 mg / ml, the loading volume is 5 ml, the ultraviolet detector detection wavelength is 220 nm, the eluent is ultrapure water, and the effluent of the 1-2 tubes (45-76 min) is collected, concentrated to an appropriate amount, and freeze-dried to obtain yak skin oligopeptide with good solubility.
[0067] The yak skin oligopeptide obtained in the present embodiment is subjected to a-glucosidase inhibitory activity determination, and the inhibition rate is 22.13%.
[0068] Example 3
[0069] As shown in the following, Figures 1-2 A preparation method of yak skin oligopeptide, the difference between the method of the present embodiment and the method of example 2 is only that the effluent of the 3-4 tubes (77-110 min) is collected to prepare yak skin oligopeptide.
[0070] The yak skin oligopeptide obtained in the present embodiment is subjected to a-glucosidase inhibitory activity determination, and the inhibition rate is 53.20%.
[0071] Example 4
[0072] As shown in the following,Figures 1-2 As shown in the figure, a preparation method of yak skin oligopeptide, the method of the embodiment is different from the method of embodiment 2 only in that the effluent of the 5th tube (111~140 min) is collected to prepare the yak skin oligopeptide.
[0073] The yak skin oligopeptide obtained in the embodiment is subjected to α-glucosidase inhibitory activity determination, and the inhibition rate is 33.86%.
[0074] As shown in the figure, F1, F2 and F3 are the yak skin oligopeptides obtained in embodiments 2-4, and it can be seen that the molecular weights of F1, F2 and F3 obtained by collecting effluents at different times are different. Figure 1 As shown in the figure, F1, F2 and F3 are the yak skin oligopeptides obtained in embodiments 2-4, and it can be seen that the molecular weights of F1, F2 and F3 obtained by collecting effluents at different times are different.
[0075] Figure 2 It can be known that after the yak skin oligopeptides (F1, F2 and F3) obtained in embodiments 2-4 are subjected to α-glucosidase inhibitory activity determination, it is found that the α-glucosidase activity of F2 is the strongest, the inhibition rate is 53.20%, and there is a significant difference between the inhibition rates of F1 and F3; F3 is the second, the inhibition rate is 33.86%, and there is a significant difference between the inhibition rates of F1 and F3.
[0076] Embodiment 5
[0077] The embodiment provides an oligopeptide RDIFL with α-glucosidase inhibitory activity, and the oligopeptide RDIFL is synthesized in the order of arginine-aspartic acid-isoleucine-phenylalanine-leucine by using a conventional solid-phase synthesis method. The oligopeptide RDIFL is detected by LC-MS / MS, and the obtained HPLC chromatogram is as shown in the figure, and the mass spectrum is as shown in the figure. Figure 3 Figure 4 The molecular weight of the synthesized oligopeptide RDIFL is 662.79 Da, and the purity is greater than 95%.
[0078] Embodiment 6
[0079] The embodiment provides an oligopeptide FRVP with α-glucosidase inhibitory activity, and the oligopeptide FRVP is synthesized in the order of phenylalanine-arginine-valine-proline by using a conventional solid-phase synthesis method. The oligopeptide FRVP is detected by LC-MS / MS, and the obtained HPLC chromatogram is as shown in the figure, and the mass spectrum is as shown in the figure. Figure 5 Figure 6 The molecular weight of the synthesized oligopeptide FRVP is 517.63 Da, and the purity is greater than 95%.
[0080] Embodiment 7
[0081] The present example provides an oligopeptide RDILF having α-glucosidase inhibitory activity. The oligopeptide RDILF is synthesized in the order of arginine-aspartic acid-isoleucine-leucine-phenylalanine by using a conventional solid-phase synthesis method. The oligopeptide RDILF is detected by LC-MS / MS, and the obtained HPLC chromatogram is shown in Figure 7 , and the mass spectrum is shown in Figure 8 . The molecular weight of the synthesized oligopeptide RDILF is 662.79 Da, and the purity is >95%.
[0082] Example 8
[0083] The present example provides an oligopeptide FDLRF having α-glucosidase inhibitory activity. The oligopeptide FDLRF is synthesized in the order of phenylalanine-aspartic acid-leucine-arginine-phenylalanine by using a conventional solid-phase synthesis method. The oligopeptide FDLRF is detected by LC-MS / MS, and the obtained HPLC chromatogram is shown in Figure 9 , and the mass spectrum is shown in Figure 10 . The molecular weight of the synthesized oligopeptide FDLRF is 696.80 Da, and the purity is >95%.
[0084] Application Example 1
[0085] The main components of the yak skin oligopeptide obtained in Examples 2-4 are detected by LC-MS / MS, and the specific process of LC-MS / MS is as follows.
[0086] (1) The yak skin oligopeptide is dissolved with the mobile phase as a sample, and then subjected to liquid phase separation and mass spectrometry. The A liquid used in the liquid phase is 0.1% v / v formic acid aqueous solution, and the B liquid is 0.1% v / v formic acid acetonitrile aqueous solution (acetonitrile concentration is 84% v / v). The liquid chromatography column is PepMap RSLC C18 (75 um x 150mm), and the column is equilibrated with 95% A liquid. The sample is loaded onto the Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by the automatic sampler, and then separated by the liquid chromatography column. The relevant liquid phase gradient is set as follows: 0 min to 50 min, B liquid linear gradient from 4% to 40%; 50 min to 60 min, B liquid linear gradient from 40% to 95%; 60 min to 65 min, B liquid maintained at 95%.
[0087] (2) The yak skin oligopeptide sample is separated by capillary high performance liquid chromatography and subjected to mass spectrometry analysis by mass spectrometer. The analysis time is 60 min. The detection mode is positive ion. The mass-to-charge ratio of the yak skin oligopeptide and the fragments of the yak skin oligopeptide is collected according to the following method: 10 fragment spectra (MS2 scan) are collected after each full scan.
[0088] (3) Database search was performed by PEAKS software, and finally 5131 polypeptide sequences were obtained, including oligopeptides RDIFL, FRVP, RDILF and FDLRF.
[0089] Application Example 2
[0090] The 5131 peptides obtained in Application Example 1 were screened for potential bioactive peptides, toxicity, water solubility, allergenicity, total average hydrophilicity and pharmacokinetics prediction. Finally, oligopeptides RDIFL, FRVP, RDILF and FDLRF were selected for verification according to the molecular docking binding energy.
[0091] As shown in Figures 11-14 , the safety and activity of the four oligopeptides RDIFL, FRVP, RDILF and FDLRF synthesized in Examples 5-8 were predicted respectively.
[0092] Toxicity and allergenicity were predicted by ToxinPred and AllerCatPro 2.0 respectively. It was predicted that RDIFL, FRVP, RDILF and FDLRF had no toxicity and allergenicity.
[0093] After pharmacokinetic evaluation of the peptides, RDIFL, FRVP, RDILF and FDLRF had good human intestinal absorption capacity.
[0094] The α-glucosidase 3D structure (PDB ID: 3A4A) was downloaded from the PDB database as the acceptor. Pymol was used for structure preprocessing, removing water, solvent molecules, other ligands and unnecessary metal ions, and adding hydrogen to the protein receptor. The 3D structure of the peptide segment was prepared using Novopro, setting Calculations for energy minimization processing and saving as an sdf format file. The format was converted to a pbd file using Open Babel, and the pdbqt file was batch-produced by Autodock Vina. AutoDock Vina 1.1.2.0 (the Scripps Research Institute, La Jolla, CA, USA) software was used for molecular docking operation, and Pymol software was used for visualization processing. The docking engine was selected as AutoDock Vina, the center point coordinates (x, y, z) = (19.3, -3.3, 21.9), and the size was set to (25.5 x 24.9 x 30.1).
[0095] The docking results are shown in Figures 11-14As shown, arginine-aspartic acid-isoleucine-phenylalanine-leucine (RDIFL) forms 6 hydrogen bonds, 1 carbon-hydrogen bond, 1 pi-pi stack with Ser240, Leu313, Arg315, Gln279, Glu277, Asp215 residues of α-glucosidase, and there are 7 interactions; phenylalanine-arginine-valine-proline (FRVP) forms 8 hydrogen bonds, 1 pi-pi stack with Asp352, Gln277, Gln279, Asp242, Lys156, Ser240, Ser241 residues of α-glucosidase, and there are 3 interactions; arginine-aspartic acid-isoleucine-leucine-phenylalanine (RDILF) forms 4 hydrogen bonds, 3 carbon-hydrogen bonds, 1 Pi-Pi stack with Arg315, Pro312, Tyr158, Gln279 residues of α-glucosidase, and there are 6 interactions; phenylalanine-aspartic acid-leucine-arginine-phenylalanine (FDLRF) forms 9 hydrogen bonds, 1 carbon-hydrogen bond, 1 T-type Pi-Pi stack with Asp242, Gln279, Asp307, Thr306, Gln353, Ser240, Lys156 residues of α-glucosidase, and there are 5 interactions.
[0096] The binding energies of acarbose and the four oligopeptides to α-glucosidase inhibition are shown in Table 1.
[0097] Table 1 Binding energies of oligopeptides to α-glucosidase inhibition
[0098]
[0099] As can be seen from Table 1, compared with acarbose, the oligopeptides of the present application have lower binding energy to α-glucosidase inhibition, and have good application prospects in the prevention and treatment of type 2 diabetes.
[0100] Application Example 3
[0101] The four oligopeptides RDIFL, FRVP, RDILF and FDLRF synthesized in Examples 5-8 were used as samples to investigate their half-inhibitory concentrations for α-glucosidase.
[0102] The in vitro inhibitory activity of α-glucosidase was determined using p-nitrophenyl-α-D-glucopyranoside (PNPG) as a substrate.
[0103] Blank group: 120 uL of PBS was mixed with 50 uL (enzyme activity was 5 U / ml) of a-glucosidase solution and placed in a 37°C water bath for 20 min, then 25 uL of PNPG solution (8 mmol) was added and incubated for 8 min, and then determined by a microplate reader.
[0104] Blank control group: 120 uL of PBS was mixed with 25 uL of PNPG solution (8 mmol), incubated for 8 min, and then determined by a microplate reader.
[0105] Sample group: 120 uL of PBS was mixed with 75 uL of sample solution (oligopeptide concentration was 500 µM~20 mM, solvent was 0.1% DMSO) and 50 uL (enzyme activity was 5 U / ml) of a-glucosidase solution, and placed in a 37°C water bath for 20 min, then 25 uL of PNPG solution (8 mmol) was added and incubated for 8 min, and then determined by a microplate reader.
[0106] Sample control group: 120 uL of PBS was mixed with 75 uL of sample solution (oligopeptide concentration was 500 µM~20 mM, solvent was 0.1% DMSO) and placed in a 37°C water bath for 20 min, then 25 uL of PNPG solution (8 mmol) was added and incubated for 8 min, and then determined by a microplate reader.
[0107] The a-glucosidase inhibition rate was calculated according to the following formula.
[0108] a-glucosidase inhibition rate (%) = [1- (S-S0) / (C-C0)] x 100
[0109] In the formula: S is the absorbance of the sample group; S0 is the absorbance of the sample control group; C is the absorbance of the blank group; C0 is the absorbance of the blank control group.
[0110] The fitting analysis was performed by Graphpad Prism 10, and the significance analysis was performed by SPSS software, and the analysis results are shown in Table 2, the IC 50 values of oligopeptides RDIFL, FRVP, RDILF, and FDLRF were 10.107 mmol / L, 13.063 mmol / L, 8.653 mmol / L, and 12.369 mmol / L, respectively, indicating that the oligopeptides had strong inhibitory ability on a-glucosidase.
[0111] Table 2 IC 50 values of active polypeptides on a-glucosidase inhibition
[0112]
[0113] Note: a, b, c, d represent significant differences between different samples.
[0114] Application Example 4
[0115] As Figures 15-17 shown, the blood glucose lowering effect evaluation was carried out on the yak skin crude peptide (powder) obtained from Example 2.
[0116] (1) Grouping and administration method: 40 SPF 4-week-old male Kunming mice weighing 21-22 g were selected and adaptively fed for one week before the experiment. The 40 mice were randomly divided into 4 groups, namely normal control group (NC group), diabetes model group (DC group), positive control group (PC group) and Example 2 group (CP group). The normal control group (NC group) was fed with basic feed, and the other 3 groups were fed with high-sugar and high-fat feed for four weeks to induce diabetes, and after four weeks, the three groups of mice were intraperitoneally injected with streptozotocin (STZ) dissolved in buffer for three consecutive days, with a dose of 50 mg / kg; while the normal control group (NC group) was intraperitoneally injected with citrate buffer. After one week, the fasting blood glucose (FBG) level of the diabetes model group (DC group), the positive control group (PC group) and the Example 2 group (CP group) mice was more than 11.1 mmol / L -1 , then the type 2 diabetes (T2DM) mice were determined. Then the normal control group and the diabetes model group: 0.2 ml of sterile normal saline was administered by gavage; the positive control group: 200 mg / Kg•BW of metformin was administered by gavage, and the Example 2 group: the yak skin crude peptide powder of Example 2 was dissolved in normal saline and administered by gavage at a dose of 200 mg / Kg•BW, for 30 days.
[0117] (2) Fasting blood glucose determination: Fasting blood glucose is a routine index for diagnosing type 2 diabetes. During the 30-day gavage period, the blood glucose level of the tail vein of all mice was determined once a week at a fixed time using a handheld blood glucose meter, and the mice were fasted for 8 hours but not watered.
[0118] (3) Oral glucose tolerance test: The oral glucose tolerance test was performed three days before the end of the experiment. The mice were fasted for 8 hours but not watered, and orally administered with glucose (2 g / Kg body weight). A handheld blood glucose meter was used to collect the tail vein blood of the mice to determine the blood glucose level at 0, 0.5, 1, 1.5 and 2 hours.
[0119] (4) Data processing: SPSS 26 software was used for one-way ANOVA, and P<0.05 was considered statistically significant.
[0120] The blood glucose lowering effect evaluation is shown in Figures 15-17 . As Figure 15It can be seen that the fasting blood glucose level of the yak skin crude peptide group of Example 2 is reduced by 15.58% compared with the diabetic model group. It shows that the yak skin crude peptide can inhibit the increase of blood glucose in diabetic mice.
[0121] Figure 16 The change of glucose tolerance in mice can be seen from the figure. Compared with the diabetic model group, the blood glucose level of the Example 2 group and the positive control group decreases faster. Figure 17 It can be seen that the Example 2 group significantly reduces the AUC value of the T2DM mouse within 120 min. The results show that the yak skin crude peptide has a good regulating effect on type 2 diabetes.
[0122] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for preparing a low molecular weight peptide from yak skin, characterized in that, The preparation method of the yak skin oligopeptide is a solid-phase synthesis method, and the yak skin oligopeptide is RDILF, wherein the amino acid sequence of the RDILF is arginine-aspartic acid-isoleucine-leucine-phenylalanine.
2. Use of a yak skin oligopeptide in the preparation of a drug for preventing and treating type 2 diabetes, characterized in that, The yak skin oligopeptide is RDILF, wherein the amino acid sequence of the RDILF is arginine-aspartic acid-isoleucine-leucine-phenylalanine.
3. A functional food, characterized by comprising the composition according to claim 1 or 2. The yak skin oligopeptide is RDILF, wherein the amino acid sequence of the RDILF is arginine-aspartic acid-isoleucine-leucine-phenylalanine.
4. A medicine, characterized in that, The yak skin oligopeptide is RDILF, wherein the amino acid sequence of the RDILF is arginine-aspartic acid-isoleucine-leucine-phenylalanine.