Background
Diabetes is a high-risk chronic disease, and in recent years, with the development of socioeconomic, the dietary structure and the life style of people change, and the incidence rate of diabetes, particularly type II diabetes, is increasing year by year. In 2030, the number of people suffering from diabetes mellitus is 4 hundred million, wherein diabetes mellitus type II accounts for the majority, and accounts for about 90%, and various clinical treatment medicines are easy to cause various side effects such as hypoglycemia, hypotension, liver injury and the like, so that the development of a novel food-borne active substance for adjuvant treatment of diabetes mellitus has a very important application prospect. Several studies have shown that bioactive peptides have an effect on improving diabetes. For example, in the research of the Wangweibo and the like, the marine collagen peptide can relieve the structural damage of islet beta cells of a rat with hyperinsulinemia, increase the secretion of particles, reduce the formation of lipid droplets and obviously improve the biological activity of insulin; it also has certain improving effect on fasting blood glucose and oral glucose tolerance.
Dipeptidyl peptidase-4 (DPP-4) is a serine protease, which can cleave Xaa-Pro or Xaa-Ala structure at the N-terminal of peptides such as hormones, and glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic hormone (GIP) can promote the secretion of insulin of the body and inhibit the secretion of glucagon in the process of diabetes, and has important effects in regulating the blood sugar balance of the human body. The DPP-4 is found to cut off two amino acids at the amino terminal of GLP-1 and GIP, so that the physiological function of the GLP-1 and the GIP is lost, and the blood sugar regulation is influenced. Therefore, inhibiting the activity of DPP-4 is beneficial to control blood sugar level, and DPP-4 inhibitors have become a hotspot for research on diabetes treatment.
At present, the traditional enzymolysis-separation-purification method is mainly adopted to screen and identify the hypoglycemic peptide, the process is complicated, the time consumption is long, a large amount of manpower and material resources are consumed, the yield is low, and certain monopeptide with strong activity is easy to miss due to the segmented activity determination method. Therefore, it is necessary to find a method for efficiently finding active peptides to realize mass production.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a hypoglycemic tetrapeptide with DPP-4 inhibitory activity and application thereof. The active peptide sieve is selected from yak type I collagen sequences, the screened peptide is obtained by solid phase synthesis, a DPP-4 and HepG2 (human hepatoma cell) cell insulin resistance model is taken as a research object, the in-vitro blood glucose reduction effect of the synthetic peptide is detected, and the result shows that the peptide has better DPP-4 inhibitory activity and IC 50 Is 0.225mg/mL, can obviously improve the insulin resistance phenomenon, promotes the absorption of glycogen, and has certain hypoglycemic effect.
The technical scheme of the invention is as follows:
a tetrapeptide having dipeptidyl peptidase-4 inhibitory activity, said tetrapeptide being abbreviated as MGPR, the amino acid sequence of said tetrapeptide being Met-Gly-Pro-Arg.
The molecular weight of the tetrapeptide is 459.56 Da.
The tetrapeptide is of a single-chain linear structure, is white powder and is easily soluble in water.
The tetrapeptide is derived from yak collagen.
Inhibitory Activity IC of the tetrapeptides on dipeptidyl peptidase-4 50 It was 0.225 mg/mL.
The tetrapeptide is chemically synthesized by a solid phase synthesis method, and the purity is more than 98%.
Further, the solid phase synthesis method comprises the following steps:
according to the characteristics of an amino acid sequence Met-Gly-Pro-Arg, a carboxyl group of Met is firstly connected with a resin in a covalent bond mode, then an amino group of Met and a carboxyl group of Gly are subjected to a glycidyl reaction, Pro, an amino group of Gly and a carboxyl group of Pro are added for a reaction after treatment, then the last Arg amino acid is added, the resin is removed after the reaction, and the MGPR is purified through high performance liquid chromatography to obtain the target polypeptide.
A pharmaceutical composition comprising said tetrapeptide having dipeptidyl peptidase-4 inhibitory activity.
An application of the tetrapeptide with the dipeptidyl peptidase-4 inhibitory activity in preparing the drugs for preventing and treating diabetes.
An application of the tetrapeptide with the dipeptidyl peptidase-4 inhibitory activity in preparing health-care food for preventing and treating diabetes.
The invention utilizes a bioinformatics method and various online active peptide databases to screen new hypoglycemic peptide from yak collagen. Firstly, performing virtual enzymolysis on yak collagen to obtain an enzymolysis peptide segment, screening unreported 2-5 peptides from the enzymolysis peptide segment, then predicting properties such as bioactivity score, molecular weight, water solubility, ADMET (absorption, distribution, metabolism, excretion and toxicity) and the like of the screened sequence, and performing molecular docking by using Discovery studio2019 software.
The result shows that the tetrapeptide MGPR has good water solubility, no toxicity, good small intestine absorption and good blood brain barrier permeability, and can be tightly combined with the key active site of DPP-4. The screened active peptide is synthesized by adopting a solid phase synthesis method, and the in-vitro DPP-4 inhibitory activity of the synthesized peptide is verified.
Advantages and technical effects of the invention
1. The invention carries out virtual screening of the directional combined polypeptide by the computer-aided technology, and compared with the traditional technology for preparing the active peptide by an enzymolysis method, the method adopted by the invention not only reduces the screening strength, shortens the research and development period, but also improves the probability of successful screening.
2. Through the search of an online database, the peptide sequence provided by the invention is not reported by a paper, and the tetrapeptide provided by the invention is a novel food-borne hypoglycemic peptide.
3. The tetrapeptide MGPR is synthesized by a solid-phase synthesis method, the in-vitro DPP-4 inhibitory activity of the synthesized peptide is detected, and the result shows that the synthesized peptide has potential blood sugar reducing capacity.
Detailed Description
The invention is described in detail below with reference to the figures and examples.
Example 1:
virtual screening of active peptides:
1. acquisition of Yak collagen sequence
Searching a collagen sequence from a yak source from a National Center for Biotechnology Information (NCBI) protein database, and finally selecting two chains of a yak type I collagen sequence: alpha-1(I) chain (Access: ELR60286.1) and alpha-2(I) chain (Access: ELR 46121.1).
2. Simulating gastrointestinal enzymolysis
The method comprises the steps of performing simulated enzymolysis on a yak collagen sequence by using ExPASY Peptidecter (https:// web. ExPASy. org/peptide _ cutter), selecting two digestive enzymes of pepsin (pH1.3) (EC 3.4.23.1) and trypsin (EC 3.4.21.4) to simulate human gastrointestinal tract digestive enzymolysis, screening 2-5 peptides from peptide segments generated by enzymolysis, comparing the peptides with an online database, screening unreported peptides, and performing next virtual screening.
3. Prediction of biological activity, water solubility, ADMET properties.
The bioactive potential of the selected active Peptide fragments was evaluated using the PeptideRanker (http:// distilldeep. ucd. ie/PeptideRanker /) program, the Peptide fragments with high scores were selected, the molecular weight and isoelectric point of the Peptide were predicted using the pI/Mw tool of the online software Expass (http:// web. Expasy. org/computer _ pI /), the net charge and hydrophobicity of the Peptide were predicted by Pepdraw (http:// www.tulane.edu/. about. biochem/WW/Pepdraw), and the water solubility of the Peptide was predicted using the "Peptide property calculator" function in the Innovagen program (http:// www.innovagen.com/proteomics-tools). ADMET properties are predicted by ADMETlab2.0(https:// admeash. scbdd. com /), and Human Intestinal Absorption (HIA), blood-brain barrier penetration (BBB) and acute oral toxicity are mainly used as analysis indexes. Finally, 5 peptide fragments GHR, GIR, PGPK, PGMK, MGPR and PGPR are selected to have better water solubility and ADMET properties, and the results are shown in table 1.
TABLE 1 bioactivity score, physicochemical and ADMET property prediction
4. Molecular docking screening DPP-4 inhibitory active peptide
The screened peptide fragment and DPP-4(PDB ID:5J3J) are subjected to molecular docking by using Discovery studio2019 software, and finally, a tetrapeptide MGPR with the best docking effect is selected. The existing research shows that DPP-4 has 3 key active pockets, the S1 active pocket is composed of Tyr547, Ser630, Tyr631, Val656, Trp659, Tyr662, Asn710, Val711 and His740, the S2 is composed of Glu205, Glu206 and Tyr662, Ser209, Arg358 and Phe357 constitute the S3 pocket, and the amino acid sites of the 3 active pockets are important targets for screening DPP-4 inhibitors. Docking results show that the tetrapeptide MGPR can be tightly combined with Ser630, His740, Tyr547 in the DPP-4S1 active pocket and Glu205 and Glu206 in the S2 pocket, form hydrogen bonds and electrostatic interaction, and can be combined with amino acid residues His126 and Arg125 of DPP-4 (FIG. 1). Finally, the in vitro activity verification is carried out on the synthesized polypeptide through the screened tetrapeptide synthesized by the solid phase.
Example 2:
artificial synthetic screened active peptide by solid phase synthesis method
The Fmoc solid-phase synthesis method is adopted, according to the characteristics of an amino acid sequence Met-Gly-Pro-Arg, the carboxyl of Met is firstly connected with a resin in a covalent bond mode, then the amino of Met and the carboxyl of Gly are subjected to a shrinking reaction, after treatment, the amino of Pro and Gly and the carboxyl of Pro are added for a reaction, then the last Arg amino acid is added, and after the reaction, the resin is cut off, so that the target polypeptide MGPR is obtained. Purification was performed by high performance liquid chromatography using a column model Kromasil C18, size 4.6 x 250mm, 5 μm, mobile phase a: acetonitrile containing 0.1% trifluoroacetic acid (TFA); mobile phase B: water with 0.1% TFA; the flow rate was 1.0mL/min, and the detection wavelength was 220 nm. The purity was required to be above 98% (as shown in fig. 2, table 2) and the structure was identified by MS (as shown in fig. 3).
TABLE 2
As shown in FIG. 3, the purity of the tetrapeptide MGPR was 98.11%, which meets the purity requirement of the synthetic peptide.
Example 3:
method for detecting in-vitro inhibitory activity of synthetic polypeptide MGPR on DPP-4
Detected using Sigma DPP-4 inhibitor screening kit (MAK 203).
Preparation of reagents
(1) Substrate solution: 200. mu.L of the crude product was diluted to 2.5mL with buffer solution and used in portions.
(2) Enzyme solution: 100 mu L of the original solution is diluted to 5mL by using a buffer solution and is subpackaged for use.
(3) Positive inhibitor (sitagliptin): 50 mu.L of the crude product is diluted to 0.5mL by using a buffer solution and is subpackaged for use.
(4) And (4) preparing a sample solution with gradient concentration by using a buffer solution.
The specific implementation steps are as follows: the reaction vessel was a black 96-well plate, 50. mu.L of the enzyme solution and 25. mu.L of the sample solution were added to the plate, the buffer solution was used for the enzyme control group, the sample solution was used for the positive inhibitor group, and the reaction was carried out at 37 ℃ for 10 min. Finally 25 μ L of substrate solution was added and the fluorescent FLU (FLU, λ ex 360/λ em 460) was measured every 1min during 15-30min after the reaction. A fluorescence value/time (min) curve is generated, and two time points (T) are selected in the linear range of the curve 1 And T 2 ) And obtaining T 1 And T 2 The slope of each hole in between. Determining the FLU (FLU) at a time 1 And an FLU 2 ) And used to determine the slope of the plot (Δ FLU/min).
Slope ═ FLU 2 –FLU 1 )/(T 2 –T 1 )=FLU/minute
Relative inhibition (%) - (control slope-experimental slope)/control slope X100%
According to the calculated result, an inhibition rate-concentration curve is made to obtain IC 50 The value is obtained.
As can be seen from FIG. 4, the tetrapeptide MGPR had better DPP-4 inhibitory activity, IC 50 The value was 0.225 mg/mL.
Example 4:
effect of the synthetic polypeptide MGPR on HepG2 cell insulin resistance model
Establishment of insulin resistance model
Collecting HepG2 cells in logarithmic growth phase, removing culture solution, washing with PBS for 1 time, subjecting the cells to trypsinization to obtain cell suspension with density of about 1 × 10 5 The cell suspension was inoculated into a 96-well plate at a concentration of 100. mu.L/well and incubated at 37 ℃ with 5% CO 2 The cell culture box is used for adherent culture for 12 hours. The culture medium is discarded, 18mmol/L glucosamine is added for induction culture for 18h, and an insulin resistance model is established.
Grouping after the model is built, and respectively adding 100 mu L of complete culture solution into a blank group (without being treated by glucosamine) and a model group (treated by glucosamine); the administration groups were each added with 100. mu.L of peptide solutions (MGPR:25, 50, 100, 200, 400. mu. mol/L) of different concentrations. As a positive control, 10. mu. mol/L rosiglitazone was used. After 24h of administration treatment, taking the supernatant, and detecting the glucose content in the supernatant by adopting a glucose oxidase-peroxidase coupling method (GOD-POD), wherein the specific operation method refers to the instruction of a glucose kit. The glucose consumption was calculated as the formula:
glucose consumption-blank glucose content-test group glucose content
The experimental result shows that the glucose uptake of model group cells treated by glucosamine for 18h is reduced by 38.4% compared with that of a blank control group, which indicates that the establishment of the insulin resistance model is successful. After 24h of tetrapeptide MGPR treatment, the glucose uptake capacity of the cells was tested, and as shown in FIG. 5, the active peptide MGPR improves the glucose uptake in a dose-dependent manner, wherein the MGPR with the concentration of 400. mu. mol/L can obviously promote the glucose uptake of model cells, and the glucose uptake is improved by 35.6% compared with that of a model group.
Finally, it is also noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications directly derivable or suggested from the present disclosure are considered to be within the scope of the present invention.