A heptapeptide that helps to reduce blood glucose and its use

Abstract

This invention belongs to the field of food health technology, specifically relating to a heptapeptide that helps lower blood sugar and its applications. The heptapeptide is ADTYNPR, with the amino acid sequence Ala-Asp-Thr-Tyr-Asn-Pro-Arg. This heptapeptide inhibits DPP-IV at IC50. 50 The value reached 245.51±13.55µg / mL, which can significantly lower blood glucose levels (P<0.001), with a blood glucose level decrease of 53.19%. It can be used to prepare health foods or medicines with significant effects.

Description

Technical Field

[0001] This invention belongs to the field of health technology, specifically relating to a heptapeptide that helps lower blood sugar and its applications. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a globally prevalent metabolic disease whose pathogenesis is closely related to insulin resistance, pancreatic β-cell dysfunction, and gut microbiota imbalance. While existing medications can effectively control blood sugar, long-term use carries risks of hypoglycemia, gastrointestinal side effects, and drug resistance.

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

[0004] Although the potential of peptides in lowering blood sugar has been preliminarily verified, related research and product development still face many challenges. For example, different small molecule peptides differ in their absorption, metabolic pathways, and blood sugar-lowering effects in vivo, requiring further research to optimize their preparation processes, improve their activity, and ensure their stability. Summary of the Invention

[0005] To address the aforementioned technical issues, this application utilizes rice protein as a raw material and establishes an efficient method for preparing rice peptides by optimizing the enzymatic hydrolysis process through protease screening, single-factor experiments, and response surface methodology. The basic nutritional components, molecular weight distribution, and amino acid composition of the rice peptides under the process conditions of this invention were analyzed. Furthermore, through simulated digestion, molecular docking, and zebrafish verification, polypeptides with high hypoglycemic activity were identified, and their functional mechanisms were preliminarily revealed, providing 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 which is: ADTYNPR(Ala-Asp-Thr-Tyr-Asn-Pro-Arg, SEQ ID NO.1).

[0007] Furthermore, the heptapeptide can be prepared by a chemical solid-phase synthesis method;

[0008] Furthermore, the heptapeptide ADTYNPR can also be isolated from rice protein hydrolysates;

[0009] Furthermore, the method for obtaining the heptapeptide ADTYNPR from rice protein hydrolysates is as follows:

[0010] Rice protein and water were mixed evenly at a weight ratio of 5-10:100. After heating to 40-50℃, 1200-2000 U of neutral protease was added per gram of rice protein, and the mixture was enzymatically hydrolyzed for 5-7 hours. After the enzymatic hydrolysis was completed, the supernatant was collected by centrifugation to obtain the rice polypeptide solution. Analysis showed that the rice polypeptide solution contained multiple peptides, including ADTYNPR.

[0011] Furthermore, the neutral protease is produced by Aspergillus oryzae, and the preparation method is disclosed in Chinese invention patent ZL201510176420.X;

[0012] Furthermore, the pH of the feed solution was adjusted to 7.0 ± 0.2 using a 7.5% sodium hydroxide aqueous solution during the enzymatic hydrolysis process;

[0013] Further, after the enzymatic hydrolysis is completed, the supernatant is collected by centrifugation and dried to obtain rice peptide powder;

[0014] Preferably, rice protein and water are stirred evenly at a weight ratio of 5-6:100, heated to 50°C, and the pH of the solution is adjusted to 7.0±0.2 with a 7.5% sodium hydroxide aqueous solution. 1900-2000 U of neutral protease (produced from Aspergillus oryzae, and the preparation method of the neutral protease is described in Example 1 of ZL201510176420.X) is added per gram of rice protein substrate, and enzymatic hydrolysis is performed for 5-6 hours.

[0015] Preferably, after enzymatic hydrolysis, the supernatant is collected by centrifugation; the supernatant is dried to obtain rice peptides (rice protein peptides).

[0016] The second technical solution provided by the present invention is a composition comprising the heptapeptide ADTYNPR described in the first technical solution;

[0017] Furthermore, the composition also contains one or more acceptable excipients.

[0018] The second technical solution provided by the present invention is the application of the heptapeptide ADTYNPR described in technical solution one or the composition described in technical solution two; particularly its application in lowering blood sugar, and more particularly its application in the preparation of health foods that help lower blood sugar, or its application in the preparation of medicines for preventing, improving or treating hyperglycemia;

[0019] The blood sugar lowering effects mentioned include, but are not limited to, the following:

[0020] (1) Improve glucose tolerance and alleviate glucose metabolism disorders;

[0021] (2) Improve insulin resistance and increase insulin sensitivity;

[0022] (3) It inhibits DPP-IV activity in the ileum and upregulates GLP-1 levels;

[0023] (4) Increase insulin secretion.

[0024] Furthermore, the heptapeptide ADTYNPR or the composition can be used alone in the preparation of the health food or medicine; it can also be used in combination with other components with hypoglycemic activity.

[0025] Furthermore, the heptapeptide ADTYNPR or the composition may be prepared into oral liquid, capsule, microcapsule powder, tablet, granule or emulsion, etc.

[0026] Beneficial effects:

[0027] This invention provides a heptapeptide ADTYNPR with hypoglycemic effect, wherein the heptapeptide ADTYNPR inhibits DPP-IV and IC50. 50 The value reached 245.51±13.55µg / mL, which can significantly lower blood glucose levels (P<0.001), with a blood glucose level decrease of 53.19%. It can be used to prepare health foods or medicines with significant effects. Attached Figure Description

[0028] Figure 1 Changes in the inhibitory activity of rice peptide DPP-IV before and after digestion

[0029] Among them, (A) the effect of simulated digestion on the DPP-IV inhibitory activity of rice peptide, with the final reaction concentration of rice peptide being 0.5 mg / mL; (B) the DPP-IV inhibitory activity of different concentrations of rice peptide (before digestion); (C) the DPP-IV inhibitory activity of rice peptide after simulated gastric digestion; and (D) the DPP-IV inhibitory activity of rice peptide after simulated gastric and intestinal digestion.

[0030] Figure 2 Molecular docking of peptides ADTYNPR (A), FKDEHQ (B), LLPQ (C), and AFEPL (D) with the DPP-IV receptor protein;

[0031] Note: The 3D pink bar model represents the peptide segment, the purple bar model represents the amino acid residues that bind to the DPP-IV active site, the yellow dashed line represents the hydrogen bond force, and the number in the middle of the yellow dashed line represents the bond length.

[0032] Figure 3 The effect of active peptides on blood glucose in zebrafish;

[0033] Note: Blank control group (Control), Model group (Model), Positive control group (Met, metformin). Detailed Implementation

[0034] The present invention will now be described through specific embodiments. All technical means not specifically described herein are methods well-known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the present invention.

[0035] The present invention will be further explained and described below through specific embodiments.

[0036] Example 1: Preparation of Rice Peptides

[0037] 1. Preparation method of rice peptides

[0038] a. Take 5g of rice protein (purchased from Xi'an Weizhen Biotechnology Co., Ltd., the quality indicators of rice protein are as follows: moisture 5.8%, crude protein 83.3%, crude fat 9.0%, ash 1.0%) and add it to 100g of water. While stirring, heat the mixture to 50℃, and then adjust the pH of the solution to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution.

[0039] b. After the liquid is stirred evenly and the temperature reaches 50℃, add 2000U of neutral protease per gram of rice protein substrate (the neutral protease is produced by Aspergillus oryzae, and the preparation method of the neutral protease is described in Example 1 of ZL201510176420.X). During the enzymatic hydrolysis process, adjust the pH of the liquid to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution and hydrolyze for 5.5 hours.

[0040] c. After enzymatic hydrolysis, the supernatant is collected after centrifugation.

[0041] d. The supernatant was freeze-dried to obtain rice enzymatic hydrolysate (rice peptide).

[0042] (2) Analysis of rice enzymatic hydrolysis products (rice peptides)

[0043] The main components, amino acid composition, and molecular weight distribution range of the rice enzymatic hydrolysate (rice peptide) obtained in step (1) were determined, and the results are shown in Tables 1-3 below:

[0044] Table 1. Basic nutritional components of rice peptides

[0045]

[0046] Table 2. Amino acid composition of rice peptides

[0047]

[0048] Table 3. Molecular weight distribution of rice peptides

[0049]

[0050] Example 2: Comparison of DPP-IV inhibitory activity between rice peptides prepared in Example 1 (before digestion) and rice peptides prepared in step 1 of Example 2 (after digestion).

[0051] 1. In vitro simulated digestion of rice peptides:

[0052] Weigh 1 g of the rice peptide prepared in Example 1, preheat it in a 37°C water bath, dissolve it in 10 mL of pepsin hydrochloric acid solution (2000 U / mL, pH 2.0), and simulate gastric digestion by shaking at 120 r / min for 2 h in a 37°C water bath. Add the digestive system volume to 200 mL, and take 100 mL of the inactivated protease from a boiling water bath as the gastric digestion product.

[0053] Subsequently, the pH was adjusted to 7.0, and trypsin (100 U / mL in the final reaction system) was added to the remaining sample to simulate intestinal digestion for 2 h. The simulation of intestinal digestion was completed after 2 h of shaking at 120 r / min in a 37°C water bath.

[0054] Samples were collected after gastric digestion and after gastrointestinal digestion, and then freeze-dried to obtain digested rice peptides.

[0055] 2. Comparison of DPP-IV inhibitory activity of rice peptides before and after digestion

[0056] The activity of DPP-IV was measured using the fluorescent substrate Gly-Pro-aminomethylcoumarin (AMC). DPP cleaves peptide bonds, releasing free AMC groups and generating fluorescence. The excitation wavelength is 350-360 nm, and the emission wavelength is 450-465 nm. The degree of inhibition of DPP-IV by different samples was calculated based on this. A DPP-IV inhibitor screening kit was used, and the specific procedure 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 well. Then, 50 μL of substrate (Gly-Pro-aminomethylcoumarin) solution was added, resulting in a final reaction concentration of 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 obtained by using buffer instead of DPP-IV were recorded as F. 空白 The results obtained by using buffer solution instead of the sample are recorded as F. 对照The formula for calculating the DPP-IV inhibition rate is as follows:

[0057]

[0058] Changes in DPP-IV inhibitory activity of rice peptides before and after in vitro simulated gastrointestinal digestion, as follows: Figure 1 As shown.

[0059] like Figure 1 As shown in Figure A, when the concentration of rice peptide in the reaction system was 0.5 mg / mL, the inhibition rate of rice peptide against DPP-IV before digestion was 43.02±1.64%. After 120 min of simulated gastric digestion, the inhibition rate of rice peptide against DPP-IV decreased from 43.02±1.64% to 32.68±1.41%, while the activity remained at 75.96% of that of rice peptide before digestion. After 120 min of simulated intestinal digestion, the inhibition rate decreased from 32.68±1.41% to 18.58±1.29%, while the activity remained at 56.85% of that of rice peptide after gastric digestion. After in vitro simulated digestion, the inhibition rate of rice peptide against DPP-IV showed a decreasing trend, with the overall activity remaining at 43.18% of that of rice peptide before digestion.

[0060] Figure 1 Figure B shows the DPP-IV inhibition rate of different concentrations of rice peptide. As can be seen from the figure, rice peptide exhibits strong DPP-IV inhibitory activity, IC50... 50 The value was 0.545 ± 0.017 mg / mL.

[0061] Figure 1 C and Figure 1 The figure shows the DPP-IV inhibition rates of different concentrations of rice peptide after gastric and intestinal digestion. As shown in the figure, the IC50 values ​​of DPP-IV inhibition rates of rice peptide after simulated gastric and intestinal digestion were 0.924±0.053 and 1.613±0.126 mg / mL, respectively.

[0062] It is evident that although the DPP-IV inhibitory activity of rice peptides decreased during digestion, it remained at a relatively high level, indicating that its active ingredients still possess a certain degree of stability in complex digestive environments.

[0063] Example 3: LC-MS / MS identification of rice peptide sequences and molecular docking screening.

[0064] 1. Identification of peptides

[0065] The rice peptides prepared in Example 1 (before digestion) and the rice peptides prepared in Example 2 (after gastrointestinal digestion) were subjected to reduction alkylation and desalting, respectively. The processed samples were analyzed by LC-MS / MS to obtain raw files of the mass spectrometry results. The peptide sequence analysis results were obtained by analysis with PEAKS Studio10.6 Denovo.

[0066] 2. Preliminary screening of peptides

[0067] The peptide list was obtained by searching the LC-MS / MS results using PEAKS Studio 10.6 Denovo software. 270 different peptide sequences were detected in the rice peptides prepared in Example 1 (before digestion), and 397 different peptide sequences were detected in the rice peptides prepared in Example 2 (after digestion). This indicates that during digestion, rice peptides are subjected to enzymatic hydrolysis by gastric and intestinal proteases, resulting in some peptides being cleaved into smaller fragments. Considering the digestive stability of peptides, this application selected peptides that remained stable both before and after digestion for further research, as shown in Table 4. Furthermore, this application also used short peptides (pentapeptides and below) with high abundance after digestion as a screening criterion, selecting 6 post-digestion peptides as research subjects (Table 5). All selected peptides were found to be derived from rice protein, not protease.

[0068] Table 4. Identification results of stable peptides before and after digestion

[0069]

[0070] Note: Area represents the peak area; a larger value indicates higher peptide abundance.

[0071] Table 5. Identification results of short peptides after digestion

[0072]

[0073] 3. Molecular docking screening of active peptides

[0074] (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. Water molecules and ligands in the DPP-IV structure were removed using Pymol software. The peptide structure with minimal energy was constructed using Chemoffice software. Then, the DPP-IV receptor protein was processed by adding hydrogen and balancing charge using AutoDock software. 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.

[0075] The docking score indicates the docking outcome; a lower score indicates more stable binding and higher potential activity. Therefore, among peptides that remain stable before and after digestion, the two peptides with the lowest docking scores, heptapeptide ADTYNPR and hexapeptide FKDEHQ (Table 6), are likely the best-performing DPP-IV inhibitors at the molecular docking level. Among short peptides with high abundance after digestion (Table 7), considering both docking score and abundance level, the tetrapeptide LLPQ and pentapeptide AFEPL may have potentially high DPP-IV inhibitory activity. Further synthesis of these four peptides will verify their in vitro DPP-IV inhibitory activity.

[0076] Table 6. Docking scores of peptides that remain stable before and after digestion

[0077]

[0078] Table 7. Docking scores of short peptides with high abundance after digestion.

[0079]

[0080] (2) The physicochemical properties of the four peptides identified by mass spectrometry and molecular docking score screening were predicted and analyzed using bioinformatics software. The results are shown in Table 8. All four peptides exhibited acidic isoelectric points, ranging from 4.00 to 5.88. The hydrophilicity index was used to characterize the hydrophobicity of bioactive peptides; a higher negative value indicates stronger hydrophilicity. ADTYNPR, FKDEHQ, and LLPQ showed some hydrophilicity, while AFEPL exhibited strong hydrophobicity. An instability index greater than 40 indicated potential instability of the peptides. ADTYNPR and FKDEHQ showed good stability. Furthermore, none of the four peptides showed potential toxicity or sensitization.

[0081] Table 8. Predictive Analysis of Physicochemical Properties of Potentially Active Peptides

[0082]

[0083] (3) Molecular docking was used to simulate the binding mode of peptides to DPP-IV, and the visualization results are as follows: Figure 2 China A Figure 2 B, Figure 2 C, Figure 2 As shown in Figure D, the binding energies of the four potential active peptides to the active site of DPP-IV are all negative (-6.590 to -9.077 kcal / mol). The binding energy of ADTYNPR is higher than that of FKDEHQ, LLPQ, and AFEPL, indicating that its complex with DPP-IV is more stable.

[0084] Furthermore, we analyzed the interactions of these peptides with the active site of DPP-IV (Table 9). The active site of DPP-IV comprises a hydrophobic pocket S1 and a charged pocket S2. S1 consists of Tyr547, Ser630, Tyr631, Trp659, Tyr662, Tyr666, Asp708, Val711, and His740. Among these residues, Ser630-Asp708-His740 form a catalytic triplet. S2 consists of Glu205, Glu206, Arg125, Ser209, Arg358, Phe357, and Asn710.

[0085] ADTYNPR forms hydrogen bond interactions with Ser630, Asp709, Asp739 at the S1 site and Arg125, Glu205, and Glu206 at the S2 site. Figure 2 (A)

[0086] Eight hydrogen bonds are formed between FKDEHQ and DPP-IV residues (Arg125, Glu205, Glu206, Val546, Gln553, Lys554, Ser630, Tyr662, His740). Figure 2 (B)

[0087] LLPQ can form four hydrogen bonds at the S1 site with Tyr547 and at the S2 site with Arg125, Glu205, and Glu206. Figure 2 (C)

[0088] AFEPL forms hydrogen bond interactions with amino acid residues Arg125, Glu205, Glu206, Lys554, and Trp629. Figure 2 (D).

[0089] These results confirm that the four peptides can effectively inhibit DPP-IV by occupying the active sites of the enzyme (S1 and S2 sites).

[0090] Table 9 Binding energies and binding sites of potential active peptides

[0091]

[0092] Example 4: Synthesis and Functional Verification of Peptides

[0093] 1. Entrust Jier Biochemical (Shanghai) Co., Ltd. to carry out chemical synthesis.

[0094] Four peptides (purity ≥ 98%) were synthesized: heptapeptide ADTYNPR, hexapeptide FKDEHQ, tetrapeptide LLPQ, and pentapeptide AFEPL.

[0095] (1) Peptides that are stable before and after in vitro digestion: heptapeptide ADTYNPR, hexapeptide FKDEHQ;

[0096] (2) Post-digestion peptides: tetrapeptide LLPQ, pentapeptide AFEPL.

[0097] 2. Verification of DPP-IV inhibitory activity of the synthesized peptides

[0098] The method is as described above.

[0099] The results are shown in Table 10. The heptapeptide ADTYNPR exhibited the strongest DPP-IV inhibitory activity, with an IC50 value of [missing value]. 50 =289.18±16.22 µmol / L, verifying the molecular docking results. The heptapeptide ADTYNPR, hexapeptide FKDEHQ, and pentapeptide AFEPL exhibited strong DPP-IV inhibitory activity, while the tetrapeptide LLPQ showed weaker DPP-IV inhibitory activity.

[0100] Table 10 DPP-IV inhibitory activity of synthetic peptides

[0101]

[0102] Example 5: Evaluation of the hypoglycemic effect of active peptides using a zebrafish hyperglycemia model

[0103] Normally developed 4-day-fiber wild-type AB strain zebrafish were selected and placed in 6-well plates, with 3 parallel wells per group and 10 zebrafish per well. Except for the blank control group (Control group), the remaining wells were used to induce hyperglycemia in the zebrafish model for 24 hours using a combination of 4% glucose and 0.5 mM alloxan. Subsequently, a model group, a positive drug control group (Met group), and a peptide intervention group were established. Specific treatments are as follows:

[0104] Control group: System water (water specifically for zebrafish farming);

[0105] Model group: 4% glucose + 0.05 mM alloxan + system water;

[0106] Positive drug control group (Met group): 4% glucose + 0.05 mM alloxan + 10 µg / mL metformin + systemic water;

[0107] Heptapeptide ADTYNPR intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL heptapeptide ADTYNPR + systemic water;

[0108] Hexapeptide FKDEHQ intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL hexapeptide FKDEHQ + systemic water;

[0109] Tetrapeptide LLPQ intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL tetrapeptide LLPQ + system water;

[0110] Pentapeptide AFEPL intervention group: 4% glucose + 0.05 mM alloxan + 5 µg / mL pentapeptide AFEPL + systemic water.

[0111] After incubation at 28℃ for 24 h, the zebrafish were washed three times with PBS in 6-well plates to remove the sugar solution from their surface. The zebrafish were then collected into 1.5 mL centrifuge tubes, with 10 zebrafish per tube and 3 tubes collected from each treatment group. After removing the water from the centrifuge tubes, 100 μL of ethanol was added, and the tubes were left at room temperature for 15 min before being transferred to an oven at 60℃ for 2 h to dry. 5 μL of ultrapure water was added to each tube, and after leaving the tubes at room temperature for 15 min, 2 μL of the tubes were taken for glucose content determination using a blood glucose meter.

[0112] The effect of active peptides on blood glucose levels in hyperglycemic zebrafish, such as Figure 3 As shown in the figure. Compared with the control group, the blood glucose level in the model group was significantly increased after 24 h of modeling with a combination of glucose and alloxan (P<0.0001), successfully establishing a zebrafish hyperglycemia model. All peptide intervention concentrations were 5 μg / mL. Compared with the model group, metformin significantly lowered blood glucose levels in hyperglycemic zebrafish (P<0.0001), and the heptapeptide ADTYNPR significantly lowered blood glucose levels (P<0.001), with a 53.19% decrease, indicating that the heptapeptide ADTYNPR has a hypoglycemic effect. After intervention with the hexapeptide FKDEHQ, blood glucose levels decreased by 20.81%, but this was not significantly different from the model group. There were no significant differences in blood glucose levels after intervention with the tetrapeptide LLPQ and the pentapeptide AFEPL.

[0113] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail 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. The application of heptapeptide ADTYNPR in the preparation of health products that assist in lowering blood sugar, or in the preparation of medicines for the prevention, improvement, or treatment of hyperglycemia, characterized in that, The heptapeptide ADTYNPR is the only active ingredient.

2. The application as described in claim 1, characterized in that, The heptapeptide ADTYNPR is prepared into oral liquid, capsule, microcapsule powder, tablet, granule or emulsion form.

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