Chloride channel inhibitors

Abstract

The application discloses a quinoa heptapeptide with a blood sugar lowering effect and application thereof, and belongs to the technical field of bioactive peptides. The amino acid sequence of the quinoa heptapeptide PSDGPGT is Pro-Ser-Asp-Gly-Pro-Gly-Thr. It is verified through animal model experiments that the quinoa heptapeptide can effectively reduce blood sugar level and improve related metabolic indexes. The application further provides a quinoa peptide rich in the peptide segment PSDGPGT. It is verified through animal model experiments that the quinoa peptide has a significant effect on blood sugar reduction, and provides a safe and effective alternative solution for the development of diabetes treatment drugs, and has good market prospects and application potential.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a quinoa heptapeptide with hypoglycemic effect and its application. Background Technology

[0002] Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia, caused by insufficient insulin secretion or defective insulin action. Long-term hyperglycemia can lead to various serious complications such as cardiovascular disease, kidney disease, and neuropathy, placing a heavy burden on global public health systems. Currently, commonly used oral hypoglycemic agents, such as alpha-glucosidase inhibitors and dipeptidyl peptidase IV inhibitors, while having some efficacy, may cause side effects such as gastrointestinal discomfort and exhibit drug resistance with long-term use. Therefore, discovering safe and effective hypoglycemic active ingredients from natural food resources has become a current research hotspot.

[0003] Bioactive peptides have shown great potential in this field due to their high activity, low toxicity, and good absorption. Their hypoglycemic mechanisms are diverse, including inhibiting key intestinal digestive enzymes (such as α-amylase and α-glucosidase), thereby slowing the rate of carbohydrate breakdown and glucose production; prolonging endogenous glucagon-like peptide-1 (GLP-1) activity by regulating intestinal hormone secretion or inhibiting related dipeptidyl peptidases; and improving insulin resistance.

[0004] Quinoa (Chenopodium quinoa Willd.), a complete protein "super grain," is a high-quality raw material for preparing bioactive peptides. Existing research has preliminarily confirmed that quinoa protein hydrolysates possess various biological activities. For example, one study used trypsin to hydrolyze quinoa protein, and the resulting peptides showed an α-glucosidase inhibition rate of 45.31%; another study prepared quinoa glycopeptides with angiotensin-converting enzyme inhibitory activity through microbial fermentation. These studies provide directions for the high-value utilization of quinoa resources.

[0005] Although there have been some reports on the hypoglycemic activity of quinoa peptides, existing technologies still have significant shortcomings. In terms of composition, most existing technologies only describe the activity of crude quinoa protein extracts or mixed polypeptide components, failing to isolate and identify a single, clearly defined peptide sequence that exerts the core hypoglycemic effect. Currently disclosed patents related to quinoa peptides are mostly compound compositions containing quinoa peptides or mixtures with broad preparation processes. For example, patent CN111000249A discloses a compound herbal oral liquid containing quinoa peptides, but its core lies in the compound formulation and does not limit or disclose the specific sequence of the quinoa peptides themselves. Patent CN202211394984.7 mainly focuses on a processing method for a prebiotic beverage containing quinoa protein peptides, with a relatively broad efficacy claim. None of these patents claim rights to a specific quinoa peptide sequence with a definite hypoglycemic effect, resulting in broad protection scopes and unclear core technical points.

[0006] Therefore, there is an urgent need in this field to target, enzymatically hydrolyze, screen, and identify one or more hypoglycemic active peptides with well-defined amino acid sequences and clear mechanisms of action from quinoa protein. This is of significant theoretical and practical value for developing novel hypoglycemic functional factors with independent intellectual property rights and for achieving intensive processing and value enhancement in the quinoa industry. Summary of the Invention

[0007] Technical Problem to be Solved: To address the aforementioned problems, the purpose of this invention is to provide a quinoa heptapeptide with hypoglycemic effects and its applications. This invention utilizes papain, neutral protease, and flavor protease to enzymatically hydrolyze black quinoa protein powder to obtain quinoa peptides. LC-MS / MS peptide mapping analysis reveals that the quinoa peptides contain the PSDGPGT peptide segment (amino acid sequence Pro-Ser-Asp-Gly-Pro-Gly-Thr). The quinoa heptapeptide PSDGPGT exhibits α-glucosidase inhibitory activity, effectively lowering blood sugar levels without significant side effects. Therefore, it can be applied to the preparation of drugs for hypoglycemia or health foods that help maintain healthy blood sugar levels.

[0008] Technical solution: A quinoa heptapeptide, wherein the quinoa heptapeptide is a quinoa peptide with the amino acid sequence PSDGPGT (Pro-Ser-Asp-Gly-Pro-Gly-Thr).

[0009] Furthermore, the preparation methods of the quinoa heptapeptide include enzymatic hydrolysis, solid-phase synthesis, or genetic engineering.

[0010] Furthermore, the solid-phase synthesis method employs the Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials and Wang resin as a solid-phase carrier. Threonine, glycine, proline, aspartic acid, serine, and proline residues are introduced sequentially to extend the peptide chain from the C-terminus to the N-terminus, thereby synthesizing the heptapeptide PSDGPGT in the solid phase.

[0011] The present invention also provides a quinoa peptide with hypoglycemic effect, wherein the quinoa peptide with hypoglycemic effect contains the above-mentioned quinoa heptapeptide PSDGPGT.

[0012] The present invention also provides a method for preparing the above-mentioned quinoa peptides, comprising the following steps:

[0013] S1. Mix black quinoa protein powder and water at a mass ratio of 1:(18-22), and adjust the pH to 7.0±0.2;

[0014] S2. Add 0.45-0.55% papain, 0.45-0.55% neutral protease and 0.45-0.55% flavor protease by weight of black quinoa protein powder and hydrolyze for 4.5-5.5 h.

[0015] S3. After the enzymatic hydrolysis is completed, inactivate the enzyme at 100 ℃ for 10 min, centrifuge at 5500-6500 r / min for 20 min, and collect the supernatant.

[0016] S4. Quinoa peptides are obtained after freeze-drying.

[0017] Furthermore, the enzyme activities of the papain, neutral protease, and flavor protease are 200 U / mg, 150 U / mg, and 60 U / mg, respectively.

[0018] The present invention also provides a composition comprising the above-mentioned quinoa heptapeptide or quinoa peptide.

[0019] Furthermore, the quinoa heptapeptide PSDGPGT or quinoa peptides containing the PSDGPGT peptide in the composition can be used as the sole active ingredient to exert a hypoglycemic effect, or it can be combined with other active ingredients that have a hypoglycemic effect.

[0020] The present invention also provides a drug with a hypoglycemic effect, the drug comprising the above-mentioned quinoa heptapeptide, quinoa peptide or a combination thereof.

[0021] The present invention also provides a health food that helps maintain healthy blood sugar levels, the health food comprising the above-mentioned quinoa heptapeptide, quinoa peptide or a combination thereof.

[0022] Furthermore, the drug is a drug for the prevention or treatment of diabetes.

[0023] Furthermore, the diabetes is type 2 diabetes.

[0024] Furthermore, the drug also comprises a pharmaceutically acceptable carrier.

[0025] Furthermore, the pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or subject.

[0026] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

[0027] Furthermore, the formulation of the drug may be, but is not limited to, an oral formulation.

[0028] Furthermore, the formulation of the drug may be, but is not limited to, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0029] Furthermore, the dosage form of the health food that helps maintain healthy blood sugar levels is a beverage, oral liquid, capsule, microcapsule powder, tablet, granule, or emulsion.

[0030] The present invention also provides the use of the above-mentioned quinoa heptapeptide, quinoa peptide or combination in the preparation of hypoglycemic drugs.

[0031] The present invention also provides the application of the above-mentioned quinoa heptapeptide, quinoa peptide or composition in the preparation of health food products that help maintain healthy blood sugar levels.

[0032] Beneficial effects:

[0033] 1. Identification and excellent stability of a highly active hypoglycemic peptide: This invention uses black quinoa protein powder as raw material. After enzymatic hydrolysis with papain, neutral protease, and flavor protease, the hypoglycemic activity of the quinoa heptapeptide PSDGPGT (amino acid sequence Pro-Ser-Asp-Gly-Pro-Gly-Thr) was identified for the first time using LC-MS / MS peptide proteography. This peptide showed no fragmentation after simulated gastrointestinal digestion and exhibited good in vivo tolerance and stability. Furthermore, molecular docking technology verified that this heptapeptide has a high binding energy to α-glucosidase, providing a clear molecular mechanism to support its hypoglycemic effect. Artificial synthesis verification showed that this single peptide can directly exert α-glucosidase inhibition and hypoglycemic functions, filling the gap in existing technologies for quinoa hypoglycemic peptides lacking a clear core sequence.

[0034] 2. Significant hypoglycemic activity, with superior effect compared to conventional positive controls: The quinoa heptapeptide PSDGPGT of this invention has been verified in vitro to have significantly better inhibitory ability against α-glucosidase than the heptapeptide PGPPGGE at concentrations of 0.01~0.1 mg / mL. In hyperglycemic zebrafish model experiments, this heptapeptide at concentrations of 10 μg / mL, 5 μg / mL and 1 μg / mL can significantly reduce blood glucose levels in the model organisms to normal levels, with hypoglycemic effects comparable to the positive control metformin. Moreover, at the same test concentration, its activity is significantly higher than that of another potential active peptide in quinoa peptides, PGPPGGE, highlighting its core hypoglycemic advantage.

[0035] 3. A convenient and efficient process for preparing quinoa peptides with a clear hypoglycemic effect: This invention establishes a one-step process for preparing quinoa peptides rich in PSDGPGT through compound enzymatic hydrolysis. Using black quinoa protein powder as raw material, the target product can be obtained by hydration at a certain ratio, compound enzymatic hydrolysis, enzyme inactivation centrifugation, and freeze drying. The process is simple to operate, mild under mild conditions, and easy to scale up for industrial application. In a hyperglycemic zebrafish model, the quinoa peptides prepared by this process can restore blood glucose levels to normal at a concentration of 200 μg / mL, and at concentrations of 50 μg / mL and above, they can significantly reduce blood glucose levels in the hyperglycemic model organisms, confirming that they have a stable and significant hypoglycemic effect.

[0036] 4. High safety, wide range of applications, and great market potential: Compared with existing clinical oral hypoglycemic drugs, which are prone to causing gastrointestinal discomfort and drug resistance with long-term use, the quinoa heptapeptide PSDGPGT and quinoa peptides rich in this peptide have the core advantages of safety and low toxicity. Both can be used as active ingredients to prepare hypoglycemic drugs. They can be used alone as the sole hypoglycemic active ingredient or in combination with other hypoglycemic ingredients. They can be developed into drugs for the prevention or treatment of type 2 diabetes, or prepared as health foods to help maintain healthy blood sugar levels. Dosage forms include oral liquids, capsules, tablets, granules, etc., providing a new, safe and effective alternative for diabetes prevention and treatment. They have good industrialization prospects and application potential in the fields of biomedicine and health foods.

[0037] 5. Achieving high-value utilization of quinoa resources, with both industrial and scientific research value: As a high-quality whole protein grain, the current development of quinoa is mostly limited to crude extracts or mixed peptides. This invention achieves the deep processing and value enhancement of quinoa protein by targeted enzymatic hydrolysis and screening to identify a single highly active hypoglycemic peptide, providing a new direction for the diversified and high-value development of the quinoa industry. Attached Figure Description

[0038] Figure 1 This is the secondary mass spectrum of the quinoa heptapeptide PSDGPGT in Example 2. The vertical axis in the figure represents intensity; y2 +-H2O represents the second dehydrated fragment ion generated by the C-terminus cleavage of the peptide; y3 + -H2O represents the third dehydrated fragment ion produced by the C-terminus cleavage of the peptide; y3 + This indicates the third fragment ion generated by the C-terminus cleavage of the peptide; y4 + -H2O represents the fourth dehydrated fragment ion generated by the C-terminus cleavage of the peptide; y4 + This indicates the fourth fragment ion generated by the cleavage of the C-terminus of the peptide.

[0039] Figure 2 This is a schematic diagram illustrating the binding interaction between quinoa heptapeptide PSDGPGT and α-glucosidase in Example 2.

[0040] Figure 3 The figure shows the α-glucosidase inhibition rate of quinoa heptapeptide PSDGPGT. The asterisk (*) in the figure indicates a significant difference compared with the PE7 group. * indicates p < 0.05, and ** indicates p < 0.01.

[0041] Figure 4 The figure shows the hypoglycemic effect of quinoa heptapeptide PSDGPGT in a hyperglycemic zebrafish model. * indicates a significant difference compared with the blank control group (NC), ** indicates p < 0.01, **** indicates p < 0.0001; # indicates a significant difference compared with the model group (MC), ## indicates p < 0.01.

[0042] Figure 5 The figure shows a comparison of the hypoglycemic effects of quinoa heptapeptides PSDGPGT and PGPPGGE. In the figure, * indicates a significant difference compared with the blank control group (NC), * means p < 0.05, **** means p < 0.0001; # indicates a significant difference compared with the model group (MC), ## means p < 0.01.

[0043] Figure 6 The figure shows the hypoglycemic effect of quinoa peptide in a hyperglycemic zebrafish model in Example 6. The asterisk (*) indicates a significant difference compared with the blank control group (NC), where * indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001; the # indicates a significant difference compared with the model group (MC), where ## indicates p < 0.01 and ### indicates p < 0.001. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0045] Black quinoa protein was purchased from Ningbo Herbes Health Technology Co., Ltd.; papain was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an enzyme activity of 200 U / mg; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd., with an enzyme activity of 150 U / mg; and flavor protease was purchased from Angel Enzyme Preparations (Yichang) Co., Ltd., with an enzyme activity of 60 U / mg.

[0046] Example 1

[0047] The preparation method of quinoa peptides includes the following steps:

[0048] S1. Mix black quinoa protein powder and water at a mass ratio of 1:20, and adjust the pH to 7.0;

[0049] S2. Add 0.5% papain, 0.5% neutral protease and 0.5% flavor protease by weight of black quinoa protein powder, and enzymatically hydrolyze them together at 50℃ for 5 h.

[0050] S3. After the enzymatic hydrolysis is completed, inactivate the enzyme at 100 ℃ for 10 min, centrifuge at 6000 r / min for 20 min, and collect the supernatant.

[0051] S4. Quinoa peptides are obtained after freeze-drying.

[0052] Example 2 Screening of active peptides

[0053] In this embodiment, the peptide segments of quinoa peptide were identified by LC-MS / MS, and the specific mechanism of action between quinoa peptide and α-glucosidase was explored using molecular docking technology. Highly efficient α-glucosidase inhibitory peptides were also screened.

[0054] 1. LC-MS / MS peptide profiling analysis

[0055] Peptide sequencing analysis of quinoa peptide samples was performed using LC-MS / MS. A C18 reversed-phase column with a pore size of 300 Å, a specification of 100 mm × 2.1 mm, and a particle size of 1.7 μm was used. Separation was performed at a flow rate of 0.3 mL / min at a column temperature of 40 ℃. Mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) formic acid. A Thermo Fisher Q Exactive high-resolution tandem mass spectrometer was used for full scan and MS2 scans in positive ionization mode. Full scan mode mass spectrometry conditions: scan range: 200-2000 m / z, resolution: 70000; MS2 mode mass spectrometry conditions: collision voltage: 30 V, resolution: 17500. Raw mass spectrometry data (raw files) were obtained and then compared with protein databases to complete the identification and analysis of quinoa peptide sequences.

[0056] Analysis of quinoa peptides revealed that they contain heptapeptides PSDGPGT and PGPPGGE, as shown in Table 1.

[0057] Table 1. Mass spectrometry results of heptapeptides PSDGPGT and PGPPGGE in quinoa

[0058] peptide sequence length Mass-to-charge ratio (m / z) Molecular weight (Da) PSDGPGT 7 630.27 630 PGPPGGE 7 610.28 610

[0059] The secondary mass spectrum of the heptapeptide PSDGPGT is shown below. Figure 1 As shown, the fragment ions of peptides include: N-terminal fragment ions (types a, b, and c) and C-terminal fragment ions (types x, y, and z). The side chains of type a, y, and z ions break to form type d, v, and w ions, respectively. In addition, there are internal ions formed by breakage at both ends, with the b and y series ions being the most common. The primary structure of the peptide can be deduced based on the b or y series fragment ions, and the molecular weight is 630 Da. Further secondary mass spectrometry analysis of the heptapeptide using source collision-induced dissociation technology confirmed its primary structure as Pro-Ser-Asp-Gly-Pro-Gly-Thr.

[0060] 2. Molecular docking

[0061] To discover bioactive peptides with hypoglycemic potential, this study screened them based on their ability to inhibit α-glucosidase. α-glucosidase is mainly located on the brush border membrane of small intestinal epithelial cells. α-glucosidase inhibitors can bind to these inhibitors without being absorbed by the small intestine, thereby rapidly inhibiting carbohydrate hydrolysis and achieving a hypoglycemic effect. Based on this mechanism, the study conducted computer-aided screening of small peptides from quinoa that may interact with α-glucosidase.

[0062] During the screening process, peptides with a PeptideRanker prediction score exceeding 0.6 were selected. Subsequently, molecular docking simulations were performed using Discovery Studio software, utilizing the α-glucosidase crystal structure (number 5ZCE) obtained from the PDB database. The specific procedures included: pretreating the receptor protein by removing water molecules and adding hydrogen atoms to define its active site region; and constructing a small peptide structure conforming to the lowest energy conformation as a ligand. The CDOCKER method was used for docking, evaluating the binding mode, binding site, and key amino acid residues between the ligand and receptor, and ranking the results based on binding free energy and the number of hydrogen bonds.

[0063] Two heptapeptides with superior binding properties were finally selected: PSDGPGT and PGPPGGE. Specific information is summarized in Table 2.

[0064] Table 2. Peptides in quinoa peptides with potential hypoglycemic activity

[0065] peptide sequence PeptideRanker score Docking energy (kcal / mol) PSDGPGT 0.61 -62.84 PGPPGGE 0.64 -55.43

[0066] Molecular docking 2D and 3D diagrams of quinoa heptapeptide PSDGPGT with α-glucosidase are shown below. Figure 2 As shown. Chemical bond analysis revealed that the heptapeptide PSDGPGT mainly binds to α-glucosidase via van der Waals forces and hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), with a docking energy of -62.84 kcal / mol. α-glucosidase forms 17 van der Waals forces with amino acid residues GLN256, ARG415, ASP60, GLN167, HIS103, ALA200, ASP327, PHE163, MET285, GLU283, PHE225, MET385, ASP382, GLY384, ILE143, PHE144, and ARG411, and 10 hydrogen bonds with ARG197, TYR63, HIS326, ASP199, PHE282, GLN328, ASP327, ASN258, GLY286, and TRP288. The molecular docking results show that the heptapeptide PSDGPGT can bind to α-glucosidase, thereby inhibiting α-glucosidase and exerting a hypoglycemic effect.

[0067] 3. Artificially synthesized peptides

[0068] We commissioned Jier Biochemical (Shanghai) Co., Ltd. to synthesize peptides PSDGPGT (PT7) and PGPPGGE (PE7) with a purity of ≥98% for subsequent functional verification.

[0069] Example 3: In vitro α-glucosidase inhibition rate of quinoa heptapeptides PSDGPGT and PGPPGGE

[0070] Heptapeptide PT7 and PE7 solutions with concentrations of 0.005, 0.01, 0.02, 0.05, and 0.1 mg / mL were prepared using PBS. The inhibition rates of heptapeptide PT7 and PE7 on α-glucosidase were determined. 25 µL of each concentration of heptapeptide PT7 and PE7 solution was mixed with 5 U / mL α-glucosidase, and then 25 µL of PBS solution was added to each well of a 96-well plate. The plate was incubated at 37 °C for 30 min. Then, 25 µL of 3 mM PNPG solution was added to each well and mixed thoroughly, and the plate was incubated at 37 °C for 30 min. Finally, 150 µL of 0.1 M Na₂CO₃ solution was added to each well to terminate the reaction, and the absorbance was immediately measured at 405 nm. The inhibition rate was calculated using the following formula:

[0071]

[0072] In the formula, A1 and As represent the absorbance of the control group (25µL α-glucosidase + 25µL PNPG + 50µL PBS) and the sample group (25µL α-glucosidase + 25µL PNPG + 25µL samples of different concentrations + 25µL PBS), respectively. A0 and Ab represent the absorbance of the blank control group (25µL PNPG + 75µL PBS) and the blank control group (25µL PNPG + 25µL samples of different concentrations + 50µL PBS), respectively.

[0073] Experimental results are as follows Figure 3 As shown in the results, at concentrations of 0.01–0.1 mg / mL, the α-glucosidase inhibition rate of PT7 was significantly higher than that of PE7, and when the concentration reached 0.1 mg / mL, the α-glucosidase inhibition rate was as high as 55.67%. α-glucosidase is a key digestive enzyme that breaks down oligosaccharides (such as maltose and sucrose) in food into monosaccharides (glucose). Inhibition of its activity slows down and delays the digestion and absorption of carbohydrates, thereby slowing down the release and absorption of glucose, helping to smooth out peak and trough increases in postprandial blood glucose. Therefore, α-glucosidase inhibitors are considered to have the potential to lower blood glucose. These experimental results indicate that PT7 has a strong α-glucosidase inhibitory ability, suggesting its potential to lower blood glucose.

[0074] Example 4: Hypoglycemic effect of quinoa heptapeptide PSDGPGT in a hyperglycemic zebrafish model

[0075] The hypoglycemic effect of heptapeptide PT7 was determined using a hyperglycemic zebrafish model. Wild-caught AB strain 4-day-old zebrafish were placed in six-well plates, with 3 parallel wells per group and 10 zebrafish per well. The intervention groups were as follows:

[0076] (1) Blank control group (NC): system water;

[0077] (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water;

[0078] (3) Metformin positive control group (PC): 333 μM alloxan + 2.67% glucose + 5 μg / mL metformin + system water;

[0079] (4) Heptapeptide PSDGPGT intervention group (PT7-10, PT7-5, PT7-1, PT7-0.1): 333 μM alloxan + 2.67% glucose + 10 / 5 / 1 / 0.1 μg / mL heptapeptide PSDGPGT + system water.

[0080] Four-day-old zebrafish were treated according to the above intervention protocol for 24 h. They were then washed three times with PBS solution to remove the sugar solution from their surface. Using a disposable dropper, the zebrafish were transferred from the six-well plate to 1.5 mL centrifuge tubes. Excess liquid was removed, and 0.1 mL of anhydrous ethanol was added. After being placed in a cool place for 15 min, the tubes were placed in an oven at 60 °C for 120 min until dried (crushed). Finally, 5 µL of ultrapure water was added to the centrifuge tubes, and the mixture was sonicated for 10 min. 2 µL of the solution was then used to measure the glucose level (S) using a glucometer.

[0081] The results are as follows Figure 4 As shown, compared with the blank control group (NC group), the blood glucose level in the model group (MC group) after 24 h of modeling with 333 μM alloxan + 2.67% glucose was significantly increased (p < 0.0001), indicating that the hyperglycemia model was successfully established. After intervention with the positive control drug 5 μg / mL metformin (PC group), there was a significant downregulation effect compared with the MC group (p < 0.01). At intervention concentrations of 10 μg / mL, 5 μg / mL, and 1 μg / mL, the heptapeptide PSDGPGT significantly downregulated blood glucose levels compared with the MC group (p < 0.01), and the downregulation was not significantly different from that in the NC group (p > 0.05), indicating that the heptapeptide PSDGPGT has a hypoglycemic effect.

[0082] Example 5: Comparison of the hypoglycemic effects of quinoa heptapeptides PSDGPGT and PGPPGGE in a hyperglycemic zebrafish model.

[0083] The hypoglycemic effects of heptapeptide PSDGPGT (PT7) and PGPPGGE (PE7) were compared using a hyperglycemic zebrafish model. Wild-caught AB strain 4-day-old zebrafish were placed in six-well plates, with 3 parallel wells per group and 10 zebrafish per well. The intervention groups were as follows:

[0084] (1) Blank control group (NC): system water;

[0085] (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water;

[0086] (3) Metformin positive control group (PC): 333 μM alloxan + 2.67% glucose + 5 μg / mL metformin + system water;

[0087] (4) Heptapeptide PSDGPGT intervention group (PT7): 333 μM alloxan + 2.67% glucose + 1 μg / mL heptapeptide PSDGPGT + system water;

[0088] (5) Heptapeptide PPGPGGE intervention group (PE7): 333 μM alloxan + 2.67% glucose + 1 μg / mL heptapeptide PPGPGGE + system water.

[0089] Four-day-old zebrafish were treated according to the above intervention protocol for 24 h. They were then washed three times with PBS solution to remove the sugar solution from their surface. Using a disposable dropper, the zebrafish were transferred from the six-well plate to 1.5 mL centrifuge tubes. Excess liquid was removed, and 0.1 mL of anhydrous ethanol was added. After being placed in a cool place for 15 min, the tubes were placed in an oven at 60 °C for 120 min until dried (crushed). Finally, 5 µL of ultrapure water was added to the centrifuge tubes, and the mixture was sonicated for 10 min. 2 µL of the solution was then used to measure the glucose level (S) using a glucometer.

[0090] The results are as follows Figure 5 As shown, the heptapeptide PSDGPGT, at a concentration of 1 μg / mL, significantly lowered blood glucose levels compared to the MC group (p < 0.01), and the reduction was not significantly different from that in the NC group (p > 0.05). However, the heptapeptide PGPPGGE, which is also present in quinoa peptides and has a strong docking interaction with α-glucosidase molecules, showed no significant difference in blood glucose levels between the MC group and the heptapeptide group at the measured dose (1 μg / mL) (p > 0.05), indicating that the heptapeptide PSDGPGT had a better hypoglycemic effect than the heptapeptide PGPPGGE.

[0091] Example 6: Hypoglycemic effect of quinoa peptide (prepared in Example 1) in a hyperglycemic zebrafish model

[0092] The hypoglycemic effect of quinoa peptides containing the heptapeptide PT7 was determined using a hyperglycemic zebrafish model. Wild AB strain 4-day-old zebrafish were placed in six-well plates, with 3 parallel wells per group and 10 zebrafish per well. The grouping interventions were as follows:

[0093] (1) Blank control group (NC): system water;

[0094] (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water;

[0095] (3) Metformin positive control group (PC): 333 μM alloxan + 2.67% glucose + 5 μg / mL metformin + system water;

[0096] (4) Quinoa peptide intervention group (QP-200, QP-100, QP-50, QP-10): 333 μM alloxan + 2.67% glucose + 200 / 100 / 50 / 10 μg / mL quinoa peptide + system water.

[0097] Four-day-old zebrafish were treated according to the above intervention protocol for 24 h. They were then washed three times with PBS solution to remove the sugar solution from their surface. Using a disposable dropper, the zebrafish were transferred from the six-well plate to 1.5 mL centrifuge tubes. Excess liquid was removed, and 0.1 mL of anhydrous ethanol was added. After being placed in a cool place for 15 min, the tubes were placed in an oven at 60 °C for 120 min until dried (crushed). Finally, 5 µL of ultrapure water was added to the centrifuge tubes, and the mixture was sonicated for 10 min. 2 µL of the solution was then used to measure the glucose level (S) using a glucometer.

[0098] The results are as follows Figure 6 As shown, compared with the blank control group (NC group), quinoa peptides at concentrations of 200 μg / mL, 100 μg / mL, and 50 μg / mL significantly lowered blood glucose levels compared with the MC group (p < 0.001, p < 0.01, p < 0.05). At an intervention dose of 200 μg / mL, blood glucose levels were no significantly different from those in the NC group (p > 0.05). This indicates that quinoa peptides containing the heptapeptide PSDGPGT have a hypoglycemic effect.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A quinoa heptapeptide, characterized in that, The quinoa heptapeptide is a quinoa peptide with the amino acid sequence PSDGPGT.

2. The quinoa heptapeptide according to claim 1, characterized in that: The preparation methods of the quinoa heptapeptide include enzymatic hydrolysis, solid-phase synthesis, or genetic engineering.

3. A quinoa peptide with hypoglycemic effect, characterized in that, The quinoa peptide with hypoglycemic effect includes the quinoa heptapeptide described in claim 1.

4. A method for preparing quinoa peptides as described in claim 3, characterized in that, Includes the following steps: S1. Mix black quinoa protein powder and water at a mass ratio of 1:(18-22), and adjust the pH to 7.0±0.2; S2. Add 0.45-0.55% papain, 0.45-0.55% neutral protease and 0.45-0.55% flavor protease by weight of black quinoa protein powder and hydrolyze for 4.5-5.5 h. S3. After the enzymatic hydrolysis is completed, inactivate the enzyme at 100 ℃ for 10 min, centrifuge at 5500-6500 r / min for 20 min, and collect the supernatant. S4. Quinoa peptides are obtained after freeze-drying.

5. A composition, characterized in that, The composition comprises the quinoa heptapeptide of claim 1 or the quinoa peptide of claim 3.

6. A drug with hypoglycemic effect, characterized in that, The drug comprises the quinoa heptapeptide of claim 1, the quinoa peptide of claim 3, or the composition of claim 5.

7. A health food product that helps maintain healthy blood sugar levels, characterized in that, The health food contains the quinoa heptapeptide of claim 1, the quinoa peptide of claim 3, or the composition of claim 5.

8. The drug according to claim 6, characterized in that, The drug is for the prevention or treatment of diabetes.

9. The medicament according to claim 8, characterized in that, The diabetes mentioned is type 2 diabetes.

10. The medicament according to claim 6, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

11. The use of the quinoa heptapeptide of claim 1, the quinoa peptide of claim 3, or the composition of claim 5 in the preparation of a hypoglycemic drug.

12. The use of the quinoa heptapeptide of claim 1, the quinoa peptide of claim 3, or the composition of claim 5 in the preparation of health foods that help maintain healthy blood sugar levels.

Images