Camel blood polypeptide and application thereof in preparation of alpha-glucosidase inhibitor

Abstract

The invention discloses a camel blood polypeptide and application thereof in preparation of an alpha-glucosidase inhibitor, a series of polypeptide products are separated from camel blood, and it is found that the camel blood polypeptide with the amino acid sequence being YFKI, DPPTMEL, DKPFGPDL or SEPSGTAFG has the remarkable alpha-glucosidase inhibitory activity; the compound can be applied to preparation of drugs for treating diseases caused by abnormal activity of alpha-glucosidase.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a camel blood polypeptide and application thereof in preparation of an alpha-glucosidase inhibitor. BACKGROUND

[0002] Obesity is a phenomenon of excessive accumulation and / or abnormal distribution of body fat and weight gain, which is a chronic metabolic disease caused by the interaction of genetic factors, environmental factors and other factors. With the development of economy and the change of lifestyle, obesity has become a global problem. Excessive obesity can easily cause complications such as type II diabetes, fatty liver, hypertension, hyperlipidemia and various tumor diseases. Diabetes is a kind of metabolic disease caused by high blood sugar and long-term high blood sugar. Diabetes can cause damage to the heart, liver, kidney and nervous system of the patient, and can also cause endocrine disorders in the body.

[0003] Alpha-glucosidase is an enzyme expressed on the brush border of the small intestine. Carbohydrates are broken down into disaccharides such as maltose during digestion in the body, and alpha-glucosidase is mainly responsible for breaking down oligosaccharides (such as maltose, sucrose) into absorbable monosaccharides (such as glucose), thereby affecting postprandial blood glucose levels. Recent studies have found that abnormal activity of alpha-glucosidase is closely related to the occurrence and development of obesity, insulin resistance and type 2 diabetes (T2DM). Alpha-glucosidase inhibitors can inhibit the activity of alpha-glucosidase, effectively delay the cleavage of disaccharides into absorbable monosaccharides, thereby controlling the absorption of carbohydrates by the human body and significantly inhibiting the excessive postprandial blood glucose, thereby effectively preventing and / or treating diabetes, obesity and the like.

[0004] At present, in the prevention and treatment of diabetes, obesity and other diseases, the alpha-glucosidase inhibitors that have been marketed mainly include acarbose, metformin hydrochloride and miglitol, etc. However, long-term use of these drugs can cause gastrointestinal reactions, intestinal flora disorders, liver damage and other side effects. Therefore, more and more scholars tend to develop safe products with alpha-glucosidase inhibitory activity.

[0005] Polypeptides are compounds composed of multiple alpha-amino acids connected by peptide bonds, which are widely sourced and have various types, and can be rapidly broken down by the digestive system protease into small molecular nutrients such as amino acids, which are safe and have no side effects. At present, a variety of natural or artificially designed polypeptide compounds with biological activity have been discovered, and polypeptide products have been widely used in medicine, health care products to improve nutrient absorption and body function. However, there are still few polypeptides reported for alpha-glucosidase inhibitory activity, especially natural active polypeptides, and it is urgent to develop natural active polypeptides with alpha-glucosidase inhibitory activity to provide a new solution for effectively preventing and / or treating diabetes, obesity and the like. SUMMARY

[0006] The present application separates a series of polypeptide products from camel blood, and finds that part of the polypeptides has significant α-glucosidase inhibitory activity, and has good application prospect in the preparation of α-glucosidase mediated disease drugs and weight loss or auxiliary blood sugar reducing health products, based on this, the present application provides a camel blood polypeptide and its application in the preparation of α-glucosidase inhibitors.

[0007] A camel blood polypeptide, the amino acid sequence of which is YFKI, DPPTMEL, DKPFGPDL or SEPSGTAFG. Alternatively, the amino acid sequence of which is YFKI or SEPSGTAFG.

[0008] The present application also provides an application of the camel blood polypeptide in the preparation of α-glucosidase inhibitors.

[0009] The present application also provides an application of the camel blood polypeptide in the preparation of a drug for preventing or treating α-glucosidase mediated diseases.

[0010] Alternatively, the disease is obesity or diabetes.

[0011] Alternatively, the diabetes is type II diabetes.

[0012] The present application also provides a drug for preventing or treating obesity or diabetes, comprising a therapeutically effective amount of the camel blood polypeptide and a pharmaceutically acceptable carrier.

[0013] The present application also provides an application of the camel blood polypeptide in the preparation of a health product for weight loss or auxiliary blood sugar reduction.

[0014] The camel blood polypeptide isolated and screened by the present application has significant α-glucosidase inhibitory activity, and can be used for the treatment of diabetes or obesity. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a total ion chromatogram (TIC) of the sample in Example 1.

[0016] Figure 2 It is a schematic diagram of the binding between the peptide and α-glucosidase.

[0017] Figure 3 It is a schematic diagram of the binding between DFF and α-glucosidase.

[0018] Figure 4 It is a schematic diagram of the binding between YFKI and α-glucosidase.

[0019] Figure 5 It is a schematic diagram of the binding between SEPSGTAFG and α-glucosidase.

[0020] Figure 6 Schematic diagram of the binding of DKPFGPDL to a-glucosidase.

[0021] Figure 7 Schematic diagram of the binding of DPPTMEL to a-glucosidase.

[0022] Figure 8 Graph of the results of the in vitro inhibition of a-glucosidase activity of YFKI (YI-4) in Example 3.

[0023] Figure 9 Graph of the results of the in vitro inhibition of a-glucosidase activity of DPPTMEL (DL-7) in Example 3.

[0024] Figure 10 Graph of the results of the in vitro inhibition of a-glucosidase activity of DKPFGPDL (DL-8) in Example 3.

[0025] Figure 11 Graph of the results of the in vitro inhibition of a-glucosidase activity of SEPSGTAFG (SG-9) in Example 3. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0028] Example 1: Identification and property prediction of camel blood polypeptides 1. Peptide sequence analysis based on LC-MS / MS Polypeptide identification is mainly achieved by the method of polypeptide extraction, reduction alkylation, desalination, and LC-MS / MS mass spectrometry detection for sequence identification of peptide samples. The specific process includes: (1) Protein extraction: centrifuge the camel blood sample at 10 kD ultrafiltration tube, 4°C, 12000 rcf for 10 min; then take the collection and wash the detection head with 2 μL of pure water for 2 times; test the sample, the loading amount is 2 μL, and the data of 280 nm is measured.

[0029] (2) Reductive alkylation: Take 50 μL of small peptide sample into a 1.5 mL EP tube, add 50 μL of 50 mM ammonium bicarbonate buffer (pH 8.0), dilute the small peptide sample to 100 μL, add 1 μL of 1 mol / L DTT (dithiothreitol) solution to it, so that the final concentration of DTT is 10 mmol / L, reduce it in a 56°C water bath for 1 h; then add 2 μL of 1 mol / L IAA (iodoacetamide) solution, so that the final concentration of IAA is 20 mmol / L, react in the dark at room temperature for 40 min; finally, add 1 μL of 1 mol / L DTT solution to neutralize the excess IAA, so that the final concentration of DTT is 10 mmol / L; and desalt with C18 stage-tip and dry at 45°C under vacuum.

[0030] (3) Liquid chromatography conditions: Use µPAC NEO HPLC Column, high throughput analysis column; mobile phase A is 0.1% FA (formic acid), mobile phase B is 0.1% FA and 80% ACN (acetonitrile); flow rate is 2.5 μL / min; analysis time of each component: 6.9 min.

[0031] The specific chromatography conditions are as follows: Table 1 Liquid chromatography analysis conditions for camel blood polypeptide sequences

[0032] (4) Mass spectrometry conditions The full scan range of mass spectrometry is 100-1500 m / z, the resolution of primary mass spectrometry is set to 240000, AGC is Custom, MaximumIT: Custom; select the parent ion with ion intensity 20 in full scan to use high-energy collision fragmentation (HCD) method to fragment, perform secondary mass spectrometry detection, AGC is Custom, MaximumIT: Custom, peptide fragment collision energy is set to 28, generate mass spectrometry detection raw data (.raw), and the results are shown in Figure 1 .

[0033] 2. Prediction of polypeptide properties by multiple databases First, use the online PeptideRanker tool (https: / / peptide ranker. com / ) to predict the properties of the polypeptide. http: / / distilldeep.ucd.ie / PeptideRanker / ) to screen potential bioactive peptides with a score >0.5 as the standard. The online databases toxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), AllertoP v.2.1 (https: / / www.ddg-pharmfac.net / allertop_test / ), innovagen (http: / / pepcalc.com / ) were used to further screen the stability of non-toxic, non-allergenic, good water solubility and potential bioactivity.

[0034] The results showed that among the 503 polypeptides, 83 peptides had a peptide ranking score >0.50, of which 80 were predicted to be non-toxic by the ToxinPred tool, 21 were allergens and 30 were non-allergens by the AllertoP v.2.1 tool, and the rest were short peptides with less than 6 amino acid residues in length. Finally, 36 potential bioactive peptides with good water solubility were screened by the Innovagen tool, including 21 short peptides, 10 medium peptides and 5 long peptides. The final screening results are shown in Table 2 below.

[0035] Table 2 Physicochemical properties and affinity energy of polypeptides in camel blood

[0036] Example 2: Analysis of the potential of polypeptides to bind to enzymes by molecular docking The sequences in Table 2 were converted to fasta format and their structures were optimized using the CHAMm force field of DS software. Since the crystal structure of Saccharomyces cerevisiae α-glucosidase was not available, the crystal structure of Saccharomyces cerevisiae iso-maltase (PDB ID: 3A4A) with high similarity retrieved from the RCSB database was used for molecular docking studies. Subsequently, the structures of the α-glucosidase receptor and the ligands (DFF, YFKI, DPPTMEL, DKPFGPDL, SEPSGTAFG) were optimized by adding nonpolar hydrogen and Gaussian charge using AutoDockTools1.5.7 software. Since there were a large number of polypeptide ligands, the cmd command line was used for batch processing of polypeptide sequences, and saved as pdbqt format. Next, the configuration file containing the docking center range was obtained and batch docking was performed using autodock vina.

[0037] The results are shown in Table 3. Figures 2 to 7 Figure 2 ​The direct visual display of the binding site and force between the peptide and the α-glucosidase, and it can be seen that the polypeptide mainly interacts with the enzyme through hydrogen bond and hydrophobic interaction; among them, DFF forms hydrogen bond with Thr310, Arg315, His280 of α-glucosidase Figure 3 ); YFKI forms hydrogen bond with Tyr158, Ser241, Asp242, His280 of α-glucosidase, and π-π interaction with Tyr158 Figure 4 ); and DPPTMEL forms hydrogen bond with Asp352, His351, Lys156 and other 6 amino acid residues Figure 7 ); DKPFGPDL Figure 5 ) and SEPSGTAFG Figure 5 ) can also stabilize the polypeptide and α-glucosidase complex structure through hydrogen bond, van der Waals force, salt bridge and other interactions. Among these amino acid residues, Tyr158, His280, Pro312, Arg315, Arg442, Asp352, Glu411, Ser240, GLu277 and Gln279 are considered to be key amino acid residues involved in the inhibition of α-glucosidase. Generally speaking, when the polypeptide interacts with the enzyme, the polypeptide can reduce the activity of the enzyme by occupying the active site of the enzyme. Therefore, the above results show that the peptide can bind to the active site of α-glucosidase, thereby reducing the activity of the enzyme.

[0038] Example 3: In vitro determination of the inhibitory activity of the peptide on α-glucosidase The peptides DFF, YFKI, DPPTMEL, DKPFGPDL and SEPSGTAFG were synthesized by solid-phase peptide synthesis method, and the synthesis process was completed by Shenzhen Bolunsi Data Biological Technology Co., Ltd.

[0039] Method for determining the inhibitory activity of α-glucosidase: A phosphate buffer solution (PBS) with a concentration of 20 mM and a pH of 6.9 was prepared, and PBS was used as a solvent to prepare 7 U / mL of α-glucosidase solution, 2.5 mM of p-nitrophenyl α-D-glucopyranoside (PNPG) solution, and polypeptide stock solution with a concentration of 100 mM, and subsequent polypeptide samples with a final concentration of 1, 4, 8, 10 mmol / L.

[0040] The experimental groups were set up as follows: a blank group (A1) consisting of a mixture of 100 μL PBS solution and 20 μL α-glucosidase solution; a blank control group (A2) consisting of 120 μL PBS solution; a sample group (B1) consisting of a mixture of 80 μL PBS solution, 20 μL α-glucosidase solution, and 20 μL sample solution; and a sample control group (B2) consisting of a mixture of 100 μL PBS solution and 20 μL sample solution. Each group had three replicate wells, and the entire reaction system was first incubated at 37°C for 15 min. Then, 20 μL PNPG was added to both the control group and the sample group, and the cells were incubated at 37°C for another 15 min. After the incubation was completed, 60 μL Na2CO3 was added to all the above groups to terminate the reaction, and the absorbance was finally measured at 405 nm.

[0041] The α-glucosidase inhibition rate (%) was calculated as follows: α-glucosidase inhibition rate (%) = [1-(B1-B2) / (A1-A2)] × 100%; In the formula, A1 represents the absorbance value of the blank group, A2 represents the absorbance value of the blank control group, B1 represents the absorbance value of the sample group, and B2 represents the absorbance value of the sample control group.

[0042] Result analysis: The results of the peptide's ability to inhibit α-glucosidase are as follows Figures 8 to 11 As shown in the figure, it can be seen that the inhibitory ability of the peptide on α-glucosidase increases with the increase of its concentration, and shows a good dose-effect relationship. Figure 8 )、DPPTMEL(DL-7)( Figure 9 )、DKPFGPDL(DL-8)( Figure 10 ) and SEPSGTAFG (SG-9) ( Figure 11 The IC 50 values ​​(representing the concentration required for 50% inhibition) of the two drugs were 1.632 mmol / L, 2.389 mmol / L, 3.141 mmol / L and 4.984 mmol / L, respectively.

[0043] The above results fully demonstrate that the peptide has a good ability to inhibit α-glucosidase activity, which is consistent with the molecular docking results.

[0044] In summary, the results of in vitro cell, enzyme activity experiment and computer simulation analysis prove that the four polypeptides Tyr-Phe-Lys-Ile (YFKI) (SEQ ID NO: 1), Asp-Pro-Pro-Thr-Met-Glu-Leu (DPPTMEL) (SEQ ID NO: 2), Asp-Lys-Pro-Phe-Gly-Pro-Asp-Leu (DKPFGPDL) (SEQ ID NO: 3), Ser-Glu-Pro-Ser-Gly-Thr-Ala-Phe-Gly (SEPSGTAFG) (SEQ ID NO: 4) all have excellent α-glucosidase inhibitory activity.

[0045] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be subject to the appended claims.

Claims

1. A camel blood polypeptide, characterized in that: Its amino acid sequence is YFKI, DPPTMEL, DKPFGPDL or SEPSGTAFG.

2. The camel blood polypeptide according to claim 1, characterized in that Its amino acid sequence is YFKI or SEPSGTAFG.

3. Use of the camel blood polypeptide as claimed in claim 1 in the preparation of an α-glucosidase inhibitor.

4. Use of the camel blood polypeptide according to claim 1 in the preparation of a medicament for preventing or treating diseases mediated by α-glucosidase.

5. The use according to claim 4, characterized in that The disease is obesity or diabetes.

6. The use according to claim 5, characterized in that The diabetes is type II diabetes.

7. A drug for preventing or treating obesity or diabetes, characterized in that: The invention comprises a therapeutically effective amount of the camel blood polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

8. Use of the camel blood polypeptide according to claim 1 in the preparation of a health product for weight loss or assisting in lowering blood sugar.

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