Donkey skin oligopeptide and application thereof

Abstract

The application discloses a donkey skin oligopeptide, and an application thereof. The amino acid sequence of the donkey skin oligopeptide is Arg-Arg-Glu-Val-Pro. The donkey skin oligopeptide disclosed by the application has alpha-glucosidase and alpha-amylase inhibitory activity, can assist in reducing blood sugar, has very important significance for developing medicines with blood sugar reducing function, and can be used for preparing alpha-glucosidase, alpha-amylase inhibitors and anti-type 2 diabetes drugs.

Description

Technical Field

[0001] This invention relates to the field of oligopeptide technology, specifically to a donkey skin oligopeptide and its applications. Background Technology

[0002] Donkey hide, from the skin of the donkey (Equus spp.), a member of the Equidae family. Equus asinus L. The skin of donkeys is rich in high-quality collagen. Donkey-hide gelatin is a traditional Chinese medicine made from donkey skin (dried or fresh) through processes such as decoction and concentration. It has the functions of nourishing blood and yin, moisturizing dryness, and stopping bleeding. It can be used to treat blood deficiency and chlorosis, dizziness and palpitations. It has been included in the "Pharmacopoeia of the People's Republic of China" (Part I) and the "Dictionary of Traditional Chinese Medicine". Therefore, the amino acid composition and amino acid sequence of donkey skin collagen may be different from collagen from other animal sources.

[0003] Donkey-hide collagen peptides are a mixture of proteins, polypeptides, oligopeptides, and amino acids (mainly oligopeptides) produced from donkey hide using enzymatic methods. Oligopeptides are small peptides with 2-10 amino acid residues. Reports indicate that oligopeptides with a molecular weight below 1000 Da and some protein molecules in donkey-hide gelatin are the main material basis for its pharmacological effects.

[0004] For example, Chinese patent document with application number CN202111417049.3 discloses a method for preparing a low-pro-inflammatory donkey-hide gelatin peptide and a product, including: (1) crushing donkey-hide gelatin blocks; (2) enzymatically hydrolyzing the crushed donkey-hide gelatin under the action of non-specific enzyme chain enzyme protein E for 3-5 hours; (3) adding protein to the glycosylation solution for enzymatic hydrolysis, so that the molecular weight of the hydrolysis product is between 1000-3000 Da, and inactivating the enzyme at high temperature; (4) freeze-drying the hydrolysis solution to obtain a pro-inflammatory donkey-hide gelatin peptide.

[0005] A method for preparing donkey skin collagen is disclosed in Chinese patent document with application number CN202011410008.7, including: (1) donkey skin pretreatment; (2) enzymatic hydrolysis membrane circulation separation; (3) decolorization and de-awakening treatment; (4) freeze drying to obtain donkey skin collagen powder with a molecular weight of 2-5kD. The triple helix structure of the collagen is not destroyed and the original biological activity is still retained.

[0006] For example, Chinese patent document with application number CN202110143314.7 discloses a method for preparing high-glutamic acid donkey collagen peptides. The method uses (1) compound enzymatic hydrolysis technology and (2) affinity chromatography to separate high-glutamic acid collagen active peptides from donkey collagen, which have strong chelating activity for ferrous ions, calcium ions and zinc ions.

[0007] However, the donkey skin collagen peptides or donkey collagen active peptides obtained by the above technical solutions are enzymatic hydrolysis products obtained by hydrolyzing collagen raw materials with active enzymes. They are mainly composed of peptides, proteins and amino acids of different molecular weights and have a certain molecular weight distribution range.

[0008] The peptides composed of enzymatic hydrolysis products have relatively similar molecular weights, making it difficult to achieve separation using a single method. Currently, peptide separation methods mainly rely on the differences in peptide properties such as molecular weight, charge, and hydrophobicity, employing a combination of multiple separation techniques, including membrane separation, chromatography, and chromatography, to achieve the desired separation.

[0009] The reported bioactivities of active oligopeptides include antioxidant, hypoglycemic, and immune-enhancing effects. For example, XU et al. (XU QY, ZHENG L, HUANG MT, et al. Exploring structural features of potentdipeptidyl peptidase Ⅳ (DPP-Ⅳ) inhibitory peptides derived from tilapia (Oreochromis niloticus) skin gelatin by an integrated approach of multivariate analysis and Gly-Pro-based peptide library[J]) hydrolyzed tilapia skin and obtained several oligopeptides with dipeptidyl peptidase-Ⅳ inhibitory activity after separation and purification. Their peptide chain structure contains -Gly-Pro- residues, which can promote the secretion of glucagon-like peptide and insulin, increase insulin levels, and lower blood sugar.

[0010] Mudgil P et al. (Mudgil P, Jobe B, Kamal H, et al. Dipeptidyl peptidese-Ⅳ, α-amylase, and angiotensin I converting enzyme inhibitory properties of novelcamel skin gelatin hydrolysates[J]) hydrolyzed camel skin collagen using three enzymatic methods (alkaline protease, neutral protease, and a mixture of alkaline protease and neutral protease) to obtain hydrolysates with dipeptidyl peptidase-Ⅳ inhibitory activity and α-amylase inhibitory activity. These hydrolysates can not only promote insulin secretion but also inhibit the breakdown of amylase and glucosidase, thus maintaining normal blood glucose levels.

[0011] Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, which is caused by insulin secretion defects or impaired biological action, or both. Hypoglycemic drugs are generally classified into three types according to their hypoglycemic mechanism: (1) stimulating insulin secretion, such as sulfonylureas; (2) increasing the utilization of glucose by peripheral tissues, such as biguanides; and (3) α-glucosidase inhibitors, such as acarbose, which is commonly used in clinical practice.

[0012] However, there are currently no reports on the separation and purification of donkey skin enzymatic hydrolysates to obtain collagen peptides with specific amino acid sequences and α-glucosidase and α-amylase inhibitory activities, which can be applied to type II diabetes. Summary of the Invention

[0013] This invention addresses the problems existing in the prior art by providing a donkey skin oligopeptide with a novel amino acid sequence. Through activity tests and animal experiments on blood sugar reduction, it was found that the donkey skin oligopeptide has inhibitory activity against α-glucosidase and α-amylase, and can help lower blood sugar.

[0014] The present invention adopts the following technical solution:

[0015] The first aspect of this invention provides a donkey skin oligopeptide with the amino acid sequence Arg-Arg-Glu-Val-Pro.

[0016] The second aspect of this invention provides the application of the above-mentioned donkey skin oligopeptide in the preparation of hypoglycemic drugs.

[0017] The third aspect of this invention provides the application of the above-mentioned donkey skin oligopeptide in the preparation of α-glucosidase and α-amylase inhibitors.

[0018] The fourth aspect of this invention provides the application of the above-mentioned donkey skin oligopeptide in the preparation of anti-type 2 diabetes drugs.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention yields three donkey skin oligopeptides with novel amino acid sequence structures: Arg-Arg-Glu-Val-Pro, Glu-Thr-Trp-Arg, and Ala-Tyr-Phe-Pro-Lys. These three donkey skin oligopeptides possess α-glucosidase and α-amylase inhibitory activities, and can assist in lowering blood sugar. This is of great significance for the development of hypoglycemic drugs and can be used to prepare α-glucosidase, α-amylase inhibitors, and anti-type II diabetes drugs. Preferably, the donkey skin oligopeptide with the amino acid sequence Arg-Arg-Glu-Val-Pro exhibits better α-glucosidase and α-amylase inhibitory effects and a better hypoglycemic effect.

[0021] 2. This invention addresses the characteristics of small molecular weight, concentrated molecular weight, and poor dispersibility of donkey skin collagen peptide mixtures prepared by enzymatic hydrolysis. A chromatographic separation method using Sephadex G-15 dextran gel resin for initial separation is employed, followed by further separation using reversed-phase high-performance liquid chromatography. Finally, LC-TOF-MS / MS peptide structure analysis identified three novel donkey skin oligopeptides. Activity tests and hypoglycemic animal experiments revealed that these three donkey skin oligopeptides possess α-glucosidase and α-amylase inhibitory activities, and can assist in lowering blood sugar. Attached Figure Description

[0022] Figure 1 The elution curves of the separated components obtained by using a dextran gel chromatography column filled with Sephadex G-15 dextran gel resin are shown. The horizontal axis represents the elution volume (mL), and the vertical axis represents the absorbance at a wavelength of 254 nm.

[0023] Figure 2 The elution curves of the separated components obtained by using a dextran gel chromatography column filled with Sephadex G-10 dextran gel resin are shown. The horizontal axis represents the elution volume (mL), and the vertical axis represents the absorbance at a wavelength of 254 nm.

[0024] Figure 3 The elution curves of the separated components obtained by using a dextran gel chromatography column filled with Sephadex G-25 dextran gel resin are shown. The horizontal axis represents the elution volume (mL), and the vertical axis represents the absorbance at a wavelength of 254 nm.

[0025] Figure 4 The image shows the Re-HPLC spectrum of component C, with the horizontal axis representing retention time (min) and the vertical axis representing the response value.

[0026] Figure 5 Mass spectra for peptide structure identification by LC-TOF-MS / MS, from top to bottom: Arg-Arg-Glu-Val-Pro, Glu-Thr-Trp-Arg, Ala-Tyr-Phe-Pro-Lys. Detailed Implementation

[0027] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] The following method was used to determine the α-glucosidase inhibitory activity: 4-nitrobenzene-α-glucopyranoside (PNPG) was used as the substrate. The reaction system consisted of 2 mL of 0.1 mol / L phosphate buffer (pH 6.8), 0.5 mL of the test sample aqueous solution, 50 µL of 1 mg / mL reduced glutathione, and 10 µL of α-glucosidase (Type I, from Saccharomyces cerevisiae, 11.4 U / mL). After shaking, the mixture was incubated at 37 °C for 10 min. Then, 200 µL of the incubated substrate PNPG (20 mmol / L) was added, and the mixture was incubated at 37 °C for 20 min. Finally, 10 mL of 0.1 mol / L Na₂CO₃ solution was added to terminate the reaction, and the absorbance was measured at a wavelength of 400 nm.

[0029] α-glucosidase inhibition rate (%) = (1-A) 样 / A 空 ) × 100%, A 空 A represents the absorbance of the blank reagent (without sample, replaced by deionized water). 样 This indicates the absorbance of the sample.

[0030] The following method was used to determine the α-amylase inhibitory activity: 1 ml of the test sample aqueous solution, 1 ml of starch azurite (prepared as a 2 mg / mL suspension using 0.05 mol / L, pH 6.9 Tris-HCl buffer and boiled for 5 min) and 50 µL of α-amylase (Type VI-B, from porcine pancrcase, 4.71 U / mL, prepared using 0.05 mol / L, pH 6.9 Tris-HCl buffer) were mixed, incubated at 38 °C for 10 min, and the reaction was terminated by adding 0.2 mL of 50% v / v glacial acetic acid aqueous solution. The mixture was centrifuged at 6000 rpm for 5 min, and the absorbance of the supernatant was measured at 595 nm.

[0031] α-Amylase inhibition rate (%) = (1-A) 样 / A 空 ) × 100%, A 空 A represents the absorbance of the blank reagent (without sample, replaced by deionized water). 样 This indicates the absorbance of the sample.

[0032] Example 1

[0033] (1) Raw material processing: Take fresh and healthy donkey skin, remove residual meat, soak it in clean water for 3-5 days, change the water 1-2 times a day, remove donkey hair, wash away impurities, cut into small pieces of about 10cm×10cm, put them into boiling water and boil for 10 minutes until the donkey skin rolls up, then take them out.

[0034] Simmering and Concentration: Add water at a ratio of 1:5 (material to liquid) and simmer for about 36 hours, adding water as needed to cover the evaporated material. Stir every 2-3 hours until the donkey hide softens. Use a tissue homogenizer to break it into a paste with no obvious particles. Heat and evaporate to concentrate the paste. 300 grams of raw donkey hide will yield 500 grams of raw donkey hide.

[0035] Enzymatic hydrolysis: 100g of the above-obtained donkey hide raw material, 0.35g of trypsin (Nanning Pangbo Bioengineering Co., Ltd., 4000U / g), and 300mL of water were placed in a 1000mL glass beaker and mixed thoroughly. The beaker was placed in a 55℃ constant temperature water bath. The pH of the enzymatic hydrolysis reaction system was adjusted to 6.0 with 1mol / L NaOH or 1mol / L HCl. The reaction solution was stirred with a stirrer at 300rpm for 8 hours. The enzyme was then inactivated at 90℃ for 20 minutes. The reaction solution was removed, cooled, and filtered. The filtrate obtained was the donkey hide enzymatic hydrolysis product (donkey hide collagen peptide mixed peptide), with a yield of 70.0%. Activity testing revealed that the inhibitory activity (IC50) of this donkey hide collagen peptide mixed peptide against α-glucosidase was [value missing]. 50 The concentration was 38.13 mg / mL, and the α-amylase inhibitory activity was 55.56 mg / mL.

[0036] Tests showed that 73% of the donkey skin collagen peptide mixture contained substances with a molecular weight of 180-1000 Da, indicating that the obtained donkey skin collagen peptide mixture was mainly oligopeptides with 2-10 amino acid residues.

[0037] (2) The filtrate was ultrafiltered using a hollow fiber polysulfone ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The filtrate was collected and concentrated by vacuum distillation and freeze-dried to obtain donkey skin collagen peptides with a molecular weight of <3000 Da.

[0038] (3) Weigh 2g of donkey skin collagen peptide, dissolve it in 2mL of water, and take 1mL of the donkey skin collagen peptide aqueous solution to load onto a dextran gel chromatography column. The dextran gel chromatography column has a specification of 2.6cm×88cm, the packing material is Sephadex G-15 dextran gel resin, the mobile phase is deionized water, the column flow rate is 1.0mL / min, and after elution, separate components A, B, C and D are obtained (the elution curves of the four separated components are shown in the figure). Figure 1 (wavelength is 254nm).

[0039] Activity assays revealed that the inhibitory activity (IC50) of isolated components A, B, C, and D on α-glucosidase was [missing value]. 50 The inhibitory activity IC50 against α-amylase was 40.15 mg / mL, 38.30 mg / mL, 22.8 mg / mL, and 60.59 mg / mL, respectively. 50 The concentrations were 62.37 mg / mL, 48.26 mg / mL, and 40.09 mg / mL, respectively, and they showed no activity.

[0040] Replace the filler with Sephadex G-10 dextran gel resin and observe its elution curve. Figure 2 It was found that the separation process could not achieve effective separation.

[0041] Replace the filler with Sephadex G-25 dextran gel resin and observe its elution curve. Figure 3 It was found that the separation process could not achieve effective separation.

[0042] (4) The fraction C with the strongest inhibitory activity of α-glucosidase and α-amylase obtained in step (3) was further separated by reversed-phase high-performance liquid chromatography (Re-HPLC). The separation conditions were as follows: chromatographic column: Angilent Eclipse XDB-C 18 Column (250×4.6mm, 5μm), mobile phase: Solution A: 0.1% v / v trifluoroacetic acid (TFA) aqueous solution, Solution B: 80% v / v acetonitrile aqueous solution, linear gradient elution: 0-5 min, 100%-80% A; 5-50 min, 80%-0% A, 50-60 min, 0% A, 60-70 min, 100% A, flow rate: 1.0 mL / min, column temperature: 30℃, detection wavelength: 340 nm. Re-HPLC chromatogram as shown. Figure 4 As shown.

[0043] like Figure 5 As shown, LC-TOF-MS / MS peptide structure analysis identified three donkey skin oligopeptides in this embodiment, with amino acid sequences of Arg-Arg-Glu-Val-Pro (RREVP), Glu-Thr-Trp-Arg (ETWR), and Ala-Tyr-Phe-Pro-Lys (AYFPK).

[0044] Activity assays revealed that the α-glucosidase inhibitory activity of the above three substances was IC50. 50 The IC50 values ​​for α-amylase inhibitory activity were 6.8 mg / mL, 9.3 mg / mL, and 12.5 mg / mL, respectively. 50 The concentrations were 11.4 mg / mL, 20.1 mg / mL, and 27.7 mg / mL, respectively.

[0045] Therefore, all three donkey skin oligopeptides can be used to prepare α-glucosidase and α-amylase inhibitors. Preferably, the donkey skin oligopeptide with the amino acid sequence Arg-Arg-Glu-Val-Pro has better inhibitory effects on α-glucosidase and α-amylase.

[0046] The mechanism of action of alpha-glucosidase inhibitors is to competitively inhibit various alpha-glucosidases located in the small intestine, slowing down the breakdown of starch into glucose, thereby reducing glucose absorption in the intestine and lowering postprandial hyperglycemia. Type 2 diabetes is caused by the glucose toxicity of postprandial hyperglycemia, which can exacerbate insulin resistance and insulin secretion defects. When pancreatic β-cell function is reduced to only about 50%, fasting blood glucose is elevated and glucose tolerance is impaired.

[0047] The mechanism of action of α-amylase inhibitors is as follows: they block the site of action of α-amylase, inhibiting its activity and preventing starch digestion; they inhibit amylase synthesis, reducing its activity in the intestine; and they inhibit amylase aggregation, preventing its activation and suppressing the rise in blood glucose levels. The role of amylase in the body is to catalyze starch compounds into glucuronic acid. Glucuronate is then broken down into glucose by uronase. Both glucuronic acid and glucose are carbohydrates that the body can directly utilize. Increased amylase activity directly affects blood glucose levels.

[0048] Based on the above research findings, the three donkey skin oligopeptides disclosed in this invention can be further used to prepare anti-type II diabetes drugs. Preferably, the donkey skin oligopeptide with the amino acid sequence Arg-Arg-Glu-Val-Pro has a better hypoglycemic effect.

[0049] The following animal experiments were conducted on the three isolated donkey skin oligopeptides, Arg-Arg-Glu-Val-Pro, Glu-Thr-Trp-Arg, and Ala-Tyr-Phe-Pro-Lys, to lower blood sugar: Arg-Arg-Glu-Val-Pro was administered at doses of 0.5 g / kg and 0.2 g / kg; Glu-Thr-Trp-Arg was administered at doses of 0.5 g / kg and 0.2 g / kg; and Ala-Tyr-Phe-Pro-Lys was administered at doses of 1.0 g / kg and 0.5 g / kg. Normal control groups and model control groups were established for all three products, and a 0.01 g / kg dose of acarbose was used as a positive control. The animals used in the donkey skin oligopeptide groups, model control group, and acarbose group were alloxan-induced diabetic mice (type II diabetes), while the animals used in the normal control group were ICR mice. The oligopeptides used in the hypoglycemic animal experiments were obtained by solid-phase synthesis, commissioned to Jier Biochemical (Shanghai) Co., Ltd.

[0050] Animals were fasted for 3-5 hours, and blood glucose levels were measured before starch administration (0 h). Each donkey skin oligopeptide group was given the same volume of the corresponding concentration of the test sample (aqueous solution), and the acarbose group was given the same volume of 0.01 g / kg acarbose. The normal control group and the model control group were given the same volume of sterile water. 15-20 minutes later, each group was given starch orally at 3-5 g / kg BW. Blood glucose levels were measured at 0.5, 1, 2, and 3 hours after starch administration. The changes in blood glucose levels and area under the curve at each time point after starch administration were observed in the model control group and each donkey skin oligopeptide group. Acarbose was used as a positive control.

[0051] The results showed that all three donkey skin oligopeptides could reduce the rate of glucose absorption and significantly improve glucose tolerance. The experimental results are shown in Tables 1, 2 and 3.

[0052] Table 1 shows the effect of Arg-Arg-Glu-Val-Pro on starch loading tolerance in alloxan-induced diabetic mice. The results in Table 1 show that the blood glucose levels in the 0.5 g / kg donkey skin oligopeptide group at 0.5, 1, 2, and 3 h after starch administration, and in the 0.2 g / kg donkey skin oligopeptide group at 0.5 h after starch administration, were significantly lower than those in the model control group (P<0.05). Compared with the model control group, the blood glucose levels in each donkey skin oligopeptide group increased more slowly and the peak value was lower; moreover, all groups showed a reduced area under the curve, exhibiting a clear dose-response relationship.

[0053]

[0054] Table 2 shows the effect of Glu-Thr-Trp-Arg on starch loading tolerance in alloxan-induced diabetic mice. The results in Table 2 show that the blood glucose levels in the 0.5 g / kg donkey skin oligopeptide group and the 0.2 g / kg donkey skin oligopeptide group were significantly lower than those in the model control group at 0.5, 1, 2, and 3 hours after starch administration (P<0.05). Compared with the model control group, the blood glucose levels in each donkey skin oligopeptide group increased more slowly and the peak value was lower; moreover, all groups showed a reduction in the area under the curve (AUC) and exhibited a clear dose-response relationship.

[0055]

[0056] Table 3 shows the effect of Ala-Tyr-Phe-Pro-Lys on starch loading tolerance in alloxan-induced diabetic mice. The results in Table 3 show that the blood glucose levels in the 1.0 g / kg donkey skin oligopeptide group and the 0.5 g / kg donkey skin oligopeptide group were significantly lower than those in the model control group at 0.5, 1, 2, and 3 hours after starch administration (P<0.05). Compared with the model control group, the blood glucose levels in each donkey skin oligopeptide group increased more slowly and the peak value was lower; moreover, all groups showed a reduced area under the curve and exhibited a clear dose-response relationship.

[0057]

Claims

1. A donkey-derived oligopeptide, characterized in that, Its amino acid sequence is Arg-Arg-Glu-Val-Pro.

2. The use of the donkey skin oligopeptide according to claim 1 in the preparation of hypoglycemic drugs.

3. The use of the donkey skin oligopeptide according to claim 1 in the preparation of anti-type II diabetes drugs.

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