Heptapeptide helpful for reducing blood sugar and application thereof

Abstract

The invention belongs to the technical field of food health, and particularly relates to a heptapeptide helpful for reducing blood sugar and application of the heptapeptide. The heptapeptide is ADTYNPR, that is, the amino acid sequence of the heptapeptide is Ala-Asp-Thr-Tyr-Asn-Pro-Arg. The heptapeptide has a DPP-IV inhibition IC50 value of 245.51 + / -13.55 g / mL, can significantly reduce a blood sugar value (Plt, 0.001) by 53.19%, can be used for preparing health food or drugs for preventing, improving or treating hyperglycemia, and has a significant effect.

Description

Technical Field

[0001] The invention belongs to the technical field of food health, and particularly relates to a heptapeptide that helps lower blood sugar and an application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a highly prevalent metabolic disease worldwide. Its onset is closely linked to insulin resistance, pancreatic beta-cell dysfunction, and intestinal flora imbalance. While existing medications can effectively control blood sugar, long-term use carries the risk of hypoglycemia, gastrointestinal side effects, and drug resistance.

[0003] Small peptides, as components with unique molecular structures and biological activities, have been extensively studied in recent years for the treatment of diabetes and metabolic diseases. Chinese invention patent application 202510122492.X, filed on January 26, 2025, discloses a sea cucumber protein-derived heptapeptide (LPPGPFP) that inhibits DPP-IV activity, thereby alleviating blood sugar levels. Chinese invention patent application 202411793504.3, filed on December 9, 2024, discloses a small peptide composition containing one or more of DQFPR, LDQFPR, and SYGLPR, which inhibits α-glucosidase and α-amylase, thereby lowering blood sugar levels.

[0004] Although the potential of peptides in lowering blood sugar has been initially validated, related research and product development still face many challenges. For example, different small molecule peptides have different absorption, metabolic pathways, and blood sugar-lowering effects in the body, and further research is urgently needed to optimize their preparation process, improve their activity, and ensure their stability. Summary of the Invention

[0005] In order to solve the above technical problems, the present application uses rice protein as raw material, optimizes the enzymatic hydrolysis process through protease screening, single-factor experiments and response surface methodology, establishes an efficient rice peptide preparation method, analyzes the basic nutritional components, molecular weight distribution and amino acid composition analysis of rice peptides under the process conditions of the present invention, and further determines the polypeptides with high hypoglycemic activity through simulated digestion, molecular docking, zebrafish verification, etc., preliminarily reveals their functional mechanism, and provides theoretical support for the in-depth development of rice peptides.

[0006] One of the technical solutions provided by the present invention is a heptapeptide, the amino acid sequence of the heptapeptide is: ADTYNPR (Ala-Asp-Thr-Tyr-Asn-Pro-Arg, SEQ ID NO. 1).

[0007] Furthermore, the heptapeptide can be prepared by chemical solid phase synthesis; Furthermore, the heptapeptide ADTYNPR can also be isolated and obtained from rice protein hydrolysate; Furthermore, the method for obtaining the heptapeptide ADTYNPR from rice protein hydrolysate is as follows: Rice protein and water are mixed uniformly in a weight ratio of 5-10:100, and after heating to 40-50° C., 1200-2000 U of neutral protease is added per gram of rice protein, and enzymolysis is carried out for 5-7 hours; after the enzymolysis is completed, the supernatant is centrifuged to obtain a rice polypeptide solution; and after analysis, the rice polypeptide solution contains multiple peptide segments including ADTYNPR; Furthermore, the neutral protease is produced from Aspergillus oryzae, and the preparation method is disclosed in Chinese invention patent ZL201510176420.X; Furthermore, during the enzymatic hydrolysis process, the pH of the feed solution was adjusted to 7.0 ± 0.2 using a 7.5% sodium hydroxide aqueous solution; Furthermore, after the enzymatic hydrolysis is completed, the supernatant is centrifuged and dried to obtain rice peptide powder; Preferably, rice protein and water are stirred in a weight ratio of 5-6:100, and after being warmed to 50° C., the pH value of the feed liquid is adjusted to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution, and 1900-2000 U of neutral protease (the neutral protease is produced from Aspergillus oryzae, and the preparation method of the neutral protease is shown in ZL201510176420.X Example 1) is added per g of rice protein substrate mass, and enzymolysis is carried out for 5-6 hours; Preferably, after the enzymatic hydrolysis is completed, the supernatant is collected by centrifugation; and the supernatant is dried to obtain rice peptide (rice protein peptide).

[0008] The second technical solution provided by the present invention is a composition comprising the heptapeptide ADTYNPR described in the first technical solution; Furthermore, the composition further comprises one or more acceptable excipients.

[0009] The second technical solution provided by the present invention is the use of the heptapeptide ADTYNPR described in the first technical solution or the composition described in the second technical solution; in particular, the use in lowering blood sugar, and more particularly, the use in preparing health foods or medicines for preventing, improving or treating hyperglycemia; The blood sugar lowering effects include but are not limited to the following: (1) Improve glucose tolerance and alleviate glucose metabolism disorders; (2) Improve insulin resistance and increase insulin sensitivity; (3) Inhibit DPP-IV activity in the ileum and upregulate GLP-1 levels; (4) Increase insulin secretion.

[0010] Furthermore, the heptapeptide ADTYNPR or the composition can be used alone in the preparation of the health food or medicine; or can be used in combination with other components having hypoglycemic activity; Furthermore, the heptapeptide ADTYNPR or the composition is prepared into the form of beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0011] Beneficial effects: The present invention provides a heptapeptide ADTYNPR with hypoglycemic effect, wherein the heptapeptide ADTYNPR has an inhibitory effect on DPP-IV IC 50 The value reached 245.51±13.55µg / mL, which can significantly lower blood sugar levels (P<0.001), with blood sugar levels falling by 53.19%. It can be used to prepare health foods or medicines for preventing, improving or treating hyperglycemia with significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Changes in DPP-IV inhibitory activity of rice peptides before and after digestion Among them, (A) Effect of simulated digestion on the DPP-IV inhibitory activity of rice peptides, the final reaction concentration of rice peptides was 0.5 mg / mL, (B) DPP-IV inhibitory activity of rice peptides at different concentrations (before digestion), (C) DPP-IV inhibitory activity of rice peptides after simulated gastric digestion, and (D) DPP-IV inhibitory activity of rice peptides after simulated gastric and intestinal digestion.

[0013] Figure 2 Molecular docking of peptides ADTYNPR (A), FKDEHQ (B), LLPQ (C), and AFEPL (D) with DPP-IV receptor protein; Note: The 3D pink stick model represents the peptide segment, the purple stick model is the amino acid residue bound to the DPP-IV active site, the yellow dotted line represents the hydrogen bond force, and the number in the middle of the yellow dotted line represents the bond length.

[0014] Figure 3 The effect of active peptides on blood glucose in zebrafish; Note: Blank control group (Control), model group (Model), positive control group (Met, metformin). DETAILED DESCRIPTION

[0015] The present invention is described below by specific embodiments. The technical means not particularly described in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material components and amounts in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0016] The present invention is further explained below through specific embodiments.

[0017] Example 1 Preparation of rice peptide 1. Preparation method of rice peptide A, get 5g rice protein (purchased from Xi'an Virgin Biotechnology Co., Ltd., rice protein quality index is as follows: moisture 5.8%, crude protein 83.3%, crude fat 9.0%, ash content 1.0%) join in 100g water, after being warming up to 50 ℃ while stirring, regulate feed liquid pH to 7.0 ± 0.2 with 7.5% sodium hydroxide aqueous solution; B, treat that feed liquid stirs, after temperature reaches 50 DEG C, (described neutral protease originates from aspergillus oryzae, the preparation method of described neutral protease is referring to ZL201510176420.X embodiment 1) adding the neutral protease of 2000U by every g rice protein substrate mass meter, the sodium hydroxide aqueous solution of enzymolysis process 7.5% regulates feed liquid pH to 7.0 ± 0.2, enzymolysis 5.5 hours; c. After the enzymatic hydrolysis is completed, the supernatant is separated by centrifuge; d. The supernatant was freeze-dried to obtain rice hydrolysate (rice peptide).

[0018] (2) Analysis of rice enzymatic hydrolysis products (rice peptides) The rice enzymatic hydrolysate (rice peptide) obtained in step (1) was subjected to determination of main components, amino acid composition and molecular weight distribution range. The results are shown in Tables 1-3 below: Table 1 Basic nutritional components of rice peptides

[0019] Table 2 Amino acid composition of rice peptides

[0020] Table 3 Molecular weight distribution of rice peptides

[0021] Example 2 Comparison of DPP-IV Inhibitory Activity of Rice Peptide Prepared in Example 1 (Before Digestion) and Rice Peptide Prepared in Example 2 Step 1 (After Digestion) 1. In vitro simulated digestion of rice peptides: Weigh 1 g of rice peptide prepared in Example 1, preheat in a 37°C water bath, and dissolve in 10 mL of pepsin hydrochloric acid solution (2000 U / mL, pH 2.0). Simulate gastric digestion by incubating in a 37°C water bath at 120 rpm for 2 hours. The digestive system volume was brought to 200 mL, and 100 mL of the protease was inactivated in a boiling water bath as the gastric digestion product.

[0022] The pH was then adjusted to 7.0, and trypsin (final reaction concentration: 100 U / mL) was added to the remaining sample to simulate intestinal digestion for 2 h. The intestinal digestion simulation was completed after 2 h of shaking in a 37°C water bath at 120 rpm.

[0023] The samples after gastric digestion and gastrointestinal digestion were collected and freeze-dried to obtain digested rice peptides.

[0024] 2. Comparison of DPP-IV inhibitory activity of rice peptides before and after digestion The activity of DPP-IV was measured using the fluorescent substrate Gly-Pro-aminomethylcoumarin (AMC). DPP cleaves peptide bonds to release free AMC groups, generating fluorescence with an excitation wavelength of 350-360 nm and an emission wavelength of 450-465 nm. This was used to calculate the degree of inhibition of DPP-IV by different samples. A DPP-IV inhibitor screening assay kit was used, and the specific operation was as follows: first, 30 μL of buffer, 10 μL of DPP-IV enzyme, and 10 μL of rice peptide sample were added to a 96-well plate and mixed. 50 μL of substrate (Gly-Pro-aminomethylcoumarin) solution was added. The final concentration of the reaction system was 100 μM. After incubation at 37°C for 30 min, the fluorescence intensity was measured and the result was recorded as F. 样品 The results were recorded as F when using buffer solution instead of DPP-IV. 空白 , using buffer solution instead of sample and recording the result as F 对照 , the calculation formula of DPP-IV inhibition rate is as follows:

[0025] Changes in DPP-IV inhibitory activity of rice peptides before and after simulated gastrointestinal digestion in vitro Figure 1 shown.

[0026] like Figure 1As shown in Figure A, when the rice peptide concentration in the reaction system was 0.5 mg / mL, the DPP-IV inhibition rate of the rice peptide before digestion was 43.02±1.64%. After 120 minutes of simulated gastric digestion, the DPP-IV inhibition rate of the rice peptide decreased from 43.02±1.64% to 32.68±1.41%, while the activity remained at 75.96% of the rice peptide before digestion. After 120 minutes of simulated intestinal digestion, the DPP-IV inhibition rate of the rice peptide decreased from 32.68±1.41% to 18.58±1.29%, while the activity remained at 56.85% of the rice peptide after gastric digestion. After simulated in vitro digestion, the DPP-IV inhibition rate of the rice peptide showed a downward trend, while the overall activity remained at 43.18% of the rice peptide before digestion.

[0027] Figure 1 Figure B shows the DPP-IV inhibition rate of rice peptides at different concentrations. As shown in the figure, rice peptides exhibit strong DPP-IV inhibitory activity, IC 50 It is 0.545±0.017 mg / mL.

[0028] Figure 1 Middle C and Figure 1 Figure D shows the DPP-IV inhibition rates of rice peptides at different concentrations after gastric and gastrointestinal digestion. The figure shows that the IC50 values ​​of DPP-IV inhibition after simulated gastric and intestinal digestion of rice peptides were 0.924±0.053 and 1.613±0.126 mg / mL, respectively.

[0029] It can be seen that although the DPP-IV inhibitory activity of rice peptide decreased during the digestion process, it still maintained a high level, indicating that its active ingredients still have a certain stability in a complex digestive environment.

[0030] Example 3 LC-MS / MS identification of rice peptide sequences and molecular docking screening 1. Identification of peptides The rice peptide samples prepared in Example 1 (before digestion) and the rice peptide samples prepared in Example 2 (after gastrointestinal digestion) were subjected to reductive alkylation and desalting, respectively. The treated samples were analyzed by LC-MS / MS to obtain raw files of the original mass spectrometry results. The raw files were analyzed by PEAKS Studio 10.6 Denovo to obtain peptide sequence analysis results.

[0031] 2. Preliminary screening of peptides Based on the LC-MS / MS results, peptide lists were retrieved using PEAKS Studio 10.6 Denovo. The rice peptides prepared in Example 1 (before digestion) yielded 270 distinct peptide sequences, while the rice peptides prepared in Example 2 (after digestion) yielded 397 distinct peptide sequences. This indicates that during digestion, rice peptides are subject to enzymatic degradation by gastric and intestinal proteases, resulting in the cleavage of some peptides into smaller fragments. Considering the digestion stability of peptides, this application selected peptides that were stable both before and after digestion for further research, as shown in Table 4. Furthermore, this application also selected six post-digestion peptides for research, using the high abundance of short peptides (pentapeptides and shorter) after digestion as a screening criterion (Table 5). All of these selected peptides were confirmed to be derived from rice protein, not protease.

[0032] Table 4 Identification results of stable peptides before and after digestion

[0033] Note: Area represents the peak area. A larger value indicates a higher abundance of the peptide.

[0034] Table 5 Identification results of short peptides after digestion

[0035] 3. Molecular docking screening of active peptides (1) The three-dimensional crystal structure of DPP-IV (PDB ID: 1WCY) was obtained from the Protein Data Bank database (http: / / www.rcsb.org / pdb) through homology comparison and retrieval. The water molecules and ligands in the DPP-IV structure were deleted using Pymol software. The energy-minimized peptide structure was constructed using Chemoffice software. The DPP-IV receptor protein was then processed using AutoDock software to add hydrogen and balance charges. The grid spacing was set to 0.375 Å in the AutoGrid program. The peptide ligand and DPP-IV receptor were docked and a stable structure was found.

[0036] The docking score represents the docking score, with lower values ​​indicating more stable binding and higher potential activity. Therefore, among the peptides that remained stable before and after digestion, the two peptides with the lowest docking scores, the heptapeptide ADTYNPR and the hexapeptide FKDEHQ (Table 6), may be the best performing DPP-IV inhibitors at the molecular docking level. Among the short peptides that were fragmented and highly abundant after digestion (Table 7), the tetrapeptide LLPQ and the pentapeptide AFEPL may have potentially high DPP-IV inhibitory activity, based on their docking scores and abundance levels. These four peptides were further synthesized to verify their in vitro DPP-IV inhibitory activity.

[0037] Table 6 Docking scores of peptides that are stable before and after digestion

[0038] Table 7 Docking scores of short peptides with high abundance after digestion

[0039] (2) The physicochemical properties of the four peptides obtained by mass spectrometry identification and molecular docking score screening were predicted and analyzed using bioinformatics software. The results are shown in Table 8. All four peptides showed acidic isoelectric points ranging from 4.00 to 5.88. The hydrophilicity index is used to characterize the hydrophobicity of bioactive peptides. The higher the negative value, the stronger the hydrophilicity. Among them, ADTYNPR, FKDEHQ and LLPQ have certain hydrophilicity, and AFEPL has strong hydrophobicity. An instability index greater than 40 indicates that the polypeptide may be unstable. ADTYNPR and FKDEHQ showed good stability. In addition, none of the four peptides had potential toxicity or sensitization.

[0040] Table 8 Prediction and analysis of physicochemical properties of potential active peptides

[0041] (3) Molecular docking was used to simulate the binding mode between the peptide and DPP-IV. The visualization results are shown in the figure below. Figure 2 Middle A, Figure 2 Middle B, Figure 2 Middle C, Figure 2 As shown in Figure D, the binding energies of the four potential active peptides to the DPP-IV active site were all negative (-6.590 to -9.077 kcal / mol). The binding energy of ADTYNPR was higher than that of FKDEHQ, LLPQ, and AFEPL, indicating that its complex with DPP-IV was more stable.

[0042] We also analyzed the interactions of these peptides with the DPP-IV active site (Table 9). The DPP-IV active site consists of a hydrophobic pocket (S1) and a charged pocket (S2). S1 is composed of Tyr547, Ser630, Tyr631, Trp659, Tyr662, Tyr666, Asp708, Val711, and His740. Among these residues, Ser630-Asp708-His740 form the catalytic triad. S2 is composed of Glu205, Glu206, Arg125, Ser209, Arg358, Phe357, and Asn710.

[0043] ADTYNPR forms hydrogen bond interactions with Ser630, Asp709, Asp739 at the S1 site and Arg125, Glu205, and Glu206 at the S2 site ( Figure 2 Middle A).

[0044] Eight hydrogen bonds are formed between FKDEHQ and DPP-IV residues (Arg125, Glu205, Glu206, Val546, Gln553, Lys554, Ser630, Tyr662, His740) Figure 2 Middle B).

[0045] LLPQ can form four hydrogen bonds with Tyr547 at the S1 site and Arg125, Glu205, and Glu206 at the S2 site ( Figure 2 Middle C).

[0046] AFEPL forms hydrogen bond interactions with amino acid residues Arg125, Glu205, Glu206, Lys554, and Trp629 ( Figure 2 Middle D).

[0047] These results confirmed that the four peptides can effectively inhibit DPP-IV by occupying the active center of the enzyme (S1 and S2 sites).

[0048] Table 9 Binding energy and binding sites of potential active peptides

[0049] Example 4 Synthesis and functional verification of peptides 1. Entrust Jier Biochemical (Shanghai) Co., Ltd. to carry out chemical synthesis Four peptides were synthesized (purity ≥ 98%), namely the heptapeptide ADTYNPR, the hexapeptide FKDEHQ, the tetrapeptide LLPQ and the pentapeptide AFEPL.

[0050] (1) Peptides that are stable before and after in vitro digestion: heptapeptide ADTYNPR and hexapeptide FKDEHQ; (2) Peptide fragments after digestion: tetrapeptide LLPQ, pentapeptide AFEPL.

[0051] 2. Verification of DPP-IV inhibitory activity of synthetic peptides The method is as described above.

[0052] The results are shown in Table 10. The heptapeptide ADTYNPR has the strongest DPP-IV inhibitory activity, IC 50=289.18±16.22 μmol / L, confirming the results of molecular docking. The heptapeptide ADTYNPR, hexapeptide FKDEHQ, and pentapeptide AFEPL showed strong DPP-IV inhibitory activity, while the tetrapeptide LLPQ showed weak DPP-IV inhibitory activity.

[0053] Table 10 DPP-IV inhibitory activity of synthetic peptides

[0054] Example 5: Evaluation of the hypoglycemic effect of active peptides in a zebrafish hyperglycemic model Normally developed 4-day-old wild-type AB zebrafish were plated in 6-well plates, with 10 zebrafish per well in three replicate wells per group. Except for the blank control group (Control group), all wells were treated with a 4% glucose and 0.5 mM alloxan solution to induce hyperglycemia for 24 hours. A model group (Model group), a positive drug control group (Met group), and a peptide intervention group were then established. Specific treatments were as follows: Blank control group (Control group): system water (special water for zebrafish breeding); Model group: 4% glucose + 0.05 mM alloxan + system water; Positive drug control group (Met group): 4% glucose + 0.05 mM alloxan + 10 µg / mL metformin + system water; Heptapeptide ADTYNPR intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL heptapeptide ADTYNPR + system water; Hexapeptide FKDEHQ intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL hexapeptide FKDEHQ + system water; Tetrapeptide LLPQ intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL tetrapeptide LLPQ + system water; Pentapeptide AFEPL intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL pentapeptide AFEPL + system water.

[0055] After incubation in a 28°C incubator for 24 h, the plates were washed three times with PBS in a 6-well plate to remove the sugar solution on the surface of the zebrafish. The zebrafish were collected into 1.5 mL centrifuge tubes, with 10 zebrafish per tube and 3 tubes collected for each treatment group. After absorbing the water in the centrifuge tubes, 100 μL of ethanol was added, and the plates were placed at room temperature for 15 min, then transferred to an oven at 60°C for 2 h for drying. 5 μL of ultrapure water was added to each tube, and after placing it at room temperature for 15 min, 2 μL of the solution was taken out and the glucose content was measured using a blood glucose meter.

[0056] Effects of active peptides on blood glucose levels in hyperglycemic zebrafish Figure 3 As shown. Compared with the control group, blood glucose levels in the model group were significantly elevated 24 hours after modeling with a combined glucose and alloxan solution (P < 0.0001), successfully establishing a hyperglycemic zebrafish model. All peptide interventions were administered at a concentration of 5 μg / mL. Compared with the model group, metformin significantly lowered blood glucose levels in hyperglycemic zebrafish (P < 0.0001). The heptapeptide ADTYNPR also significantly lowered blood glucose levels (P < 0.001), with a 53.19% decrease, demonstrating that the heptapeptide ADTYNPR has a hypoglycemic effect. Hexapeptide FKDEHQ intervention decreased blood glucose levels by 20.81%, but this was not significantly different from the model group. Interventions with the tetrapeptide LLPQ and the pentapeptide AFEPL did not result in significant differences in blood glucose levels.

[0057] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A heptapeptide that helps lower blood sugar, characterized in that: The heptapeptide is ADTYNPR, that is, the amino acid sequence is Ala-Asp-Thr-Tyr-Asn-Pro-Arg.

2. A composition comprising the heptapeptide ADTYNPR, characterized in that The composition further comprises one or more auxiliary materials.

3. Use of the heptapeptide according to claim 1 or the composition according to claim 2 in the preparation of health foods or medicines for preventing, improving or treating hyperglycemia.

4. The use according to claim 3, characterized in that The heptapeptide ADTYNPR or the composition is used alone in the preparation of the health food or medicine, or is used in combination with other components having hypoglycemic activity.

5. The use according to claim 3, characterized in that The heptapeptide ADTYNPR or the composition is prepared into the form of beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

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