A mixed type alpha-glucosidase inhibitor hippocampus peptide, its preparation method and application

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a mixed alpha-glucosidase inhibitory hippocampus peptide, a preparation method and application thereof. The present application comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1, and is prepared from hippocampus, by adopting a double-enzyme synergistic hydrolysis method and combining with an optimized process to prepare an alpha-glucosidase inhibitory peptide and application thereof. The present application significantly improves the polypeptide yield and enhances the inhibitory activity on alpha-glucosidase, and provides a new technical path for high-value utilization of marine biological resources and development of natural hypoglycemic functional components.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a hybrid inhibitory α-glucosidase activity-inhibiting hippocampal peptide, its preparation method, and its application. Background Technology

[0002] Diabetes is one of the fastest-growing chronic diseases globally, characterized by abnormally high blood sugar levels. Human glucose is primarily produced by the hydrolysis of carbohydrates by α-amylase and α-glucosidase; inhibiting the activity of these two enzymes can help control blood sugar levels. α-glucosidase is located at the brush border of small intestinal epithelial cells; its inhibitors can bind to the enzyme without being absorbed by the small intestine, rapidly blocking carbohydrate digestion and absorption, and alleviating postprandial hyperglycemia symptoms. Long-term use of drug-based inhibitors such as acarbose can easily cause bloating and abdominal pain. The development of natural, low-toxicity alternatives has become a trend, and food-derived α-glucosidase inhibitory peptides are highly favored due to their safety, low cost, and strong enzyme affinity.

[0003] Seahorse is a traditional and precious Chinese medicinal herb with a medicinal history of over a thousand years, first recorded in the *Compendium of Materia Medica*. The swollen-bellied seahorse is the largest seahorse species, exceeding 30cm in length and weighing up to 30g. It is mainly produced along the coast of Australia and New Zealand and is high in protein, rich in aromatic, heterocyclic, and acidic essential amino acids. Peptides extracted from seahorse tissue have attracted attention due to their diverse biological activities and potential therapeutic effects. Chinese patent CN119978061A discloses two novel bioactive peptides from the Chinese herbal seahorse with potent antioxidant and α-glucosidase inhibitory activities. However, this invention does not specify the type of seahorse and only preliminarily clarifies the activities of the two peptides. It does not delve into the type of inhibition, stability, and in vivo hypoglycemic mechanism, and lacks systematic process optimization and mechanism of action explanation. Therefore, further research and development are needed on the technology for preparing peptides with α-glucosidase inhibitory activity from the swollen-bellied seahorse. Summary of the Invention

[0004] The technical problem to be solved by this invention is that the technology for preparing peptides with α-glucosidase inhibitory activity from bloated seahorses still needs further research and development.

[0005] To address the aforementioned problems, this invention provides a hybrid α-glucosidase-inhibiting hippocampal peptide, its preparation method, and its applications. Using puffed hippocampus as raw material, the invention employs a dual-enzyme synergistic hydrolysis combined with optimized processes to prepare the α-glucosidase-inhibiting peptide, and its applications. This invention significantly improves peptide yield and enhances α-glucosidase inhibitory activity, providing a new technical pathway for the high-value utilization of marine biological resources and the development of natural hypoglycemic functional components.

[0006] To achieve the above objectives, the present invention is implemented through the following technical means: a hybrid inhibitory α-glucosidase activity bloated hippocampal peptide, comprising a polypeptide with an amino acid sequence as shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] PGIGFPGPT.

[0009] The preparation method of the expanded hippocampal peptide powder containing the above-mentioned mixed-type inhibitory α-glucosidase activity of expanded hippocampal peptide includes the following steps:

[0010] (1) The dried seahorse was pulverized by a pulverizer to obtain seahorse powder; the pulverized seahorse powder was accurately weighed, deionized water was added, the pH was adjusted to 7.0, animal protease at a mass ratio of 2.5-3.5% was added, and enzymatic hydrolysis was carried out in a constant temperature water bath at 55 °C for 2.5-3.5 h; after the enzyme was inactivated by high temperature, it was cooled to room temperature; then the pH was adjusted to 2.0, pepsin at a mass ratio of 2.5-3.5% was added, and enzymatic hydrolysis was continued in a constant temperature water bath at 37 °C for 1.5-2.5 h; after the enzymatic hydrolysis was completed, the enzyme was inactivated by high temperature and cooled to room temperature; at present, the enzymatic hydrolysis of protein is mild, has few by-products, and can retain the nutritional value of the hydrolysate. From the perspective of protein hydrolysis effect and hydrolysate activity, the dual-enzyme synergistic hydrolysis method usually has higher hydrolysis efficiency and better product bioactivity than the single-enzyme hydrolysis method.

[0011] (2) Centrifuge and collect the supernatant to obtain the hydrolysate of the bloated hippocampus;

[0012] (3) After filtering the above enzymatic hydrolysate through a microporous membrane, place it in an ultrafiltration system and fractionate it through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain the fraction with Mw < 3 kDa. Freeze-drying this fraction yields expanded hippocampal peptide powder. The function of microporous membrane filtration is to clarify and remove impurities: further remove fine suspended particles remaining in the supernatant after centrifugation to ensure the clarity of the sample; protect the ultrafiltration components: prevent large particles from clogging the membrane pores during subsequent ultrafiltration processes, thereby reducing membrane fouling, extending the service life of the ultrafiltration membrane, and maintaining a high membrane flux.

[0013] Furthermore, the dried seahorse from step (1) is pulverized using a pulverizer until it can all pass through an 80-mesh sieve. Passing through an 80-mesh sieve increases the specific surface area, promoting full contact between the enzyme and the substrate, thereby improving the enzymatic hydrolysis efficiency.

[0014] Further, in step (1), deionized water is added at a ratio of 1:20.

[0015] Further, step (1) involves inactivating the enzyme at high temperature by heating at 100 °C for 15 min after the enzymatic hydrolysis is completed.

[0016] Furthermore, the centrifugation parameters in step (2) are 10000 × g for 15 min at 4 °C. The high centrifugal force of 10000 × g can effectively settle small suspended particles, unhydrolyzed macromolecular proteins and colloidal impurities in the system, and significantly improve the clarity of the supernatant.

[0017] Furthermore, in step (3), the pore size of the microporous membrane is 0.22 μm. 0.22 μm is the industry gold standard pore size for removing bacteria. This step can physically retain the vast majority of bacteria and fungi in the system, effectively preventing microbial growth and avoiding peptide putrefaction or decomposition by microorganisms during the subsequent time-consuming ultrafiltration process.

[0018] A method for preparing a hybrid inhibitor of α-glucosidase activity from bloated hippocampal peptides, which is synthesized artificially.

[0019] The above-mentioned mixed-type α-glucosidase-inhibiting hippocampal peptides are used in the preparation of hypoglycemic products or α-glucosidase-inhibiting products.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) This invention uses a dual-enzyme synergistic hydrolysis technology to prepare hippocampal peptides, which significantly improves the peptide yield and enzymatic hydrolysis yield. The preparation process is green and environmentally friendly. The resulting expanded hippocampal peptide powder has high α-glucosidase inhibitory activity and excellent thermal stability, acid-base stability and gastrointestinal digestion resistance. It can effectively delay starch digestion in vivo, providing a new way for the high-value utilization of expanded hippocampus.

[0022] (2) A novel highly active peptide, PGIGFPGPT (P39), with a molecular weight of 841.96 Da, was screened and identified. The peptide was confirmed to be non-toxic and non-sensitizing, exhibiting significant α-glucosidase inhibitory activity both in vitro and in vivo. Kinetic studies clarified its mechanism of action: PGIGFPGPT is a mixed-type inhibitor. Attached Figure Description

[0023] Figure 1 The figure shows the inhibition rate of each isolated component against α-glucosidase. Different lowercase letters (ad) in the figure indicate significant differences between groups (p < 0.05).

[0024] Figure 2 It is the half-inhibition concentration IC50 50 value.

[0025] Figure 3 This represents the effect of temperature on the inhibition rate of α-glucosidase. The same letter (a) in the figure indicates no significant difference between groups (p < 0.05).

[0026] Figure 4 This represents the effect of pH on the inhibition rate of α-glucosidase. The same letter (a) in the figure indicates no significant difference between groups (p < 0.05).

[0027] Figure 5 It is an in vitro simulation of gastrointestinal digestion results.

[0028] Figure 6 This describes the effect of HAPH-1 on lowering blood glucose levels in vivo. Figure A shows the glucose tolerance curves of mice after gavage administration of starch; Figure B shows the glucose tolerance curves of mice after gavage administration of maltose; Figure C shows the area under the blood glucose curve (AUC) of the starch loading test; and Figure D shows the area under the blood glucose curve (AUC) of the maltose loading test. Different lowercase letters (a-e) in the figures indicate significant differences between groups (p < 0.05).

[0029] Figure 7 The results are from the LC-MS / MS identification of the HAPH-1 component.

[0030] Figure 8 It is the IC of peptide segments 50 value.

[0031] Figure 9 This study investigated the effect of the peptide PGIGFPGPT on postprandial blood glucose levels in mice. Figure A shows the glucose tolerance curves of mice after gavage administration of starch; Figure B shows the glucose tolerance curves of mice after gavage administration of maltose; Figure C shows the area under the blood glucose curve (AUC) for the starch loading test; and Figure D shows the area under the blood glucose curve (AUC) for the maltose loading test. Different lowercase letters (ab) in the figures indicate significant differences between groups (p < 0.05).

[0032] Figure 10 This is a kinetic analysis of the inhibition of α-glucosidase by the peptide PGIGFPGPT. Figure A shows the relationship between reaction rate and enzyme concentration; Figure B is a Lineweaver-Burk double reciprocal plot used to determine the type of inhibition. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0035] Example 1:

[0036] A method for preparing a swelling-inducing hippocampal peptide powder includes the following steps:

[0037] 1. Preparation of α-glucosidase inhibitory peptide from distended hippocampus:

[0038] The dried abdominal hippocampus was pulverized using a grinder until it could all pass through an 80-mesh sieve. The resulting powder was stored at -20 °C for later use. Accurately weigh the dried abdominal hippocampus powder and dissolve it in deionized water at a ratio of 1:20 (w / v). Adjust the pH to 7.0 with 1 mol / L NaOH, add 3% (w / w) animal protease, and hydrolyze in a 55 °C water bath for 3 h. After hydrolysis, inactivate the enzyme at 100 °C for 15 min, and then cool to room temperature. Next, adjust the pH to 2.0 with 1 mol / L HCl, add 3% (w / w) pepsin, and continue hydrolysis in a 37 °C water bath for 2 h. After hydrolysis, inactivate the enzyme at 100 °C for 15 min, cool to room temperature, and centrifuge at 10000 × g for 15 min at 4 °C. Collect the supernatant to obtain the abdominal hippocampus protein hydrolysates (HAPH).

[0039] 2. Ultrafiltration separation of α-glucosidase inhibitory peptides from distended hippocampus:

[0040] The enzymatic hydrolysate was filtered through a 0.22 μm microporous membrane and then placed in an ultrafiltration system for fractionation, passing sequentially through ultrafiltration membranes with molecular weight cutoffs of 5 kDa and 3 kDa. Based on the molecular weight range, three fractions were obtained: Mw < 3 kDa (HAPH-1), 3 kDa < Mw < 5 kDa (HAPH-2), and Mw > 5 kDa (HAPH-3). Each fraction was collected, freeze-dried, and its α-glucosidase inhibition rate was determined.

[0041] Effect verification:

[0042] 1. The method for determining the yield of polypeptides in this invention is as follows:

[0043] Take 2 mL of the enzymatic hydrolysate and mix it with 10% TCA solution. Let it stand for 10 min, centrifuge at 4000 r / min for 15 min, and then take the supernatant. Make up to 50 mL with 5% TCA. Take 3 mL of the above solution, add 2 mL of biuret reagent, mix well, and let it stand for 10 min. Measure the absorbance at 540 nm and plot a standard curve. The peptide yield is calculated using the following formula:

[0044] .

[0045] 2. The method for determining the α-glucosidase inhibition rate of this invention is as follows:

[0046] Take 200 μL of sample and add 200 μL of α-glucosidase (0.2 U / mL). Mix the solution well and incubate at 37℃ for 5 min. Then add 200 μL of 5mM p-nitrobenzene-α-glucosidase solution (pNPG) and 0.1 M phosphate buffer (pH 6.8) to a final volume of 2 mL and incubate at 37℃ for 20 min. Finally, add 200 μL of 0.4 M sodium carbonate to stop the reaction and measure the absorbance at 405 nm. The α-glucosidase inhibition rate is calculated using the following formula:

[0047] .

[0048] Where: A1: Sample absorbance value (sample + α-glucosidase + pNPG);

[0049] A2: Absorbance value of blank sample group (sample + PBS + pNPG);

[0050] A3: Absorbance value of blank group (PBS + α-glucosidase + pNPG);

[0051] A4: Absorbance value of blank control group (PBS+pNPG).

[0052] 3. Stability Study:

[0053] (1) Thermal stability: 5 mg / mL solution of HAPH-1 lyophilized powder was prepared and incubated in water baths at 20 ℃, 40 ℃, 60 ℃, 80 ℃ and 100 ℃ for 2 h. After treatment, the solution was cooled to room temperature and the α-glucosidase inhibition rate was measured.

[0054] (2) pH stability: Weigh HAPH-1 lyophilized powder to prepare a 5 mg / mL solution, adjust the pH of the sample solution to 3.0, 5.0, 7.0, 9.0 and 11.0 respectively, let it stand at room temperature for 2 h, adjust the pH to 7.0 and then measure the α-glucosidase inhibition rate.

[0055] (3) Gastrointestinal digestive stability: HAPH-1 lyophilized powder was weighed and prepared into a 5 mg / mL solution. An equal volume of artificial gastric juice (prepared by dissolving 1 g pepsin in 100 mL of distilled water and adjusting the pH to 2.0 with 1 mol / L HCl) was added. The solution was incubated in a 37℃ water bath for 2 h, and samples were taken every 30 min. After enzyme inactivation, the α-glucosidase inhibition rate was measured. After the simulated gastric digestion was completed, the digestive juice was adjusted to pH 7.5 with NaOH, and trypsin (enzyme to sample mass ratio of 1:25, w / w) was added. The digestion was continued in a 37℃ water bath for 2 h, and samples were taken every 30 min. After enzyme inactivation, the α-glucosidase inhibition rate was measured.

[0056] 4. Effect of α-glucosidase inhibitory peptide from distended hippocampus on postprandial blood glucose:

[0057] Effects of HAPH-1 on postprandial blood glucose levels in mice:

[0058] Mice were fed under standard laboratory conditions (25±1℃) and after 7 days of acclimatization, they were randomly divided into 10 groups for starch or maltose loading tests. The groups were as follows: (1) Starch control group (starch 1 g / kg + physiological saline), (2) Starch positive control group (starch 1 g / kg + acarbose 10 mg / kg), (3-5) HAPH-1 low, medium, and high dose groups (starch 1 g / kg + HAPH-1 100, 200, and 400 mg / kg), (6) Maltose control group (maltose 2 g / kg + physiological saline), (7) Maltose positive control group (maltose 2 g / kg + acarbose 10 mg / kg), (8-10) Maltose HAPH-1 low, medium, and high dose groups (maltose 2 g / kg + HAPH-1 100, 200, and 400 mg / kg). All mice were fasted for 12 h before the test, but were allowed free access to water. After fasting, mice in the corresponding groups were administered acarbose or HAPH-1 by gavage. Fifteen minutes later, the mice were administered starch or maltose by gavage. Subsequently, blood samples were collected from the tail vein of the mice at 0, 30, 60, 90, 120, and 180 minutes using a glucometer, and blood glucose curves were plotted and the area under the curve was calculated.

[0059] result:

[0060] 1. The peptide yield of the enlarged hippocampus protein hydrolysate in Example 1 was measured to be 86.89%, and the α-glucosidase inhibition rate was 63.92%. This indicates that the dual-enzyme synergistic hydrolysis system has extremely high degradation efficiency for hippocampal protein and confirms that enlarged hippocampal protein contains abundant natural hypoglycemic precursors.

[0061] 2. This invention uses 3 kDa and 5 kDa ultrafiltration membranes to separate HAPH. The inhibition rates of each separated component on α-glucosidase are as follows: Figure 1 As shown, when the peptide concentration was 5 mg / mL, HAPH-1 (Mw < 3 kDa) inhibited α-glucosidase by 75.2 ± 0.4%, with a half-maximal inhibitory concentration (IC50) of 1 / 2. 50 The value was 3.29 mg / mL ( Figure 2 This demonstrates that ultrafiltration fractionation can effectively enrich small molecule peptides with α-glucosidase inhibitory activity, and that low molecular weight peptides exhibit superior inhibitory activity.

[0062] 3. For example Figures 3-5 As shown, the α-glucosidase inhibition rate of HAPH-1 did not change significantly at temperatures ranging from 20 to 100℃ and pH ranges from 3 to 11. Furthermore, after 5 h of in vitro simulated gastrointestinal digestion treatment, its α-glucosidase inhibition rate remained at a high level, indicating that HAPH-1 has high thermal stability, acid-base stability, and gastrointestinal digestibility.

[0063] 4. To study the effect of HAPH-1 in lowering blood glucose levels in vivo, changes in blood glucose levels were measured after mice were administered starch or maltose by gavage. The inhibitory effect of HAPH-1 was evaluated by comparing the blood glucose curves and the area under the curve. Figure 6 The results in Figure A showed that postprandial blood glucose levels in mice initially increased, reaching a maximum within 0.5 h, followed by a decrease in blood glucose concentration over time, returning to initial levels within 3 h. After gavage administration of starch, HAPH-1 inhibited the increase in postprandial blood glucose levels; compared with the control group, HAPH-1 significantly reduced the area under the blood glucose curve (p<0.05) in a concentration-dependent manner, indicating that HAPH-1 can inhibit α-glucosidase activity. The trend observed after gavage administration of maltose was similar to that of starch. These results suggest that HAPH-1 has the potential to regulate starch digestion and postprandial blood glucose levels.

[0064] Example 2:

[0065] 1. Identification of the α-glucosidase inhibitory peptide sequence in the distended hippocampus by LC-MS / MS:

[0066] After desalting, the peptide sample was centrifuged and dried, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and bottled for online LCMS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 Å) at an appropriate volume, followed by a 20 μL wash for desalting. The sample was then desalted and retained on the pre-column before separation on a C18 reversed-phase column (AcclaimPepMap RSLC, 75 μm × 25 cm C18 - 2 μm 100 Å). The gradient used was a 60-minute increase in mobile phase B (80% acetonitrile, 0.1% formic acid) from 5% to 38%. Mass spectrometry was performed using a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a nano-spray Nano Flex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 320 °C.

[0067] 2. Prediction of the properties of α-glucosidase inhibitory peptides in distended hippocampus:

[0068] The potential bioactivity of the α-glucosidase inhibitory peptide from the distended hippocampus was predicted using PeptideRanker (PeptideRanker). http: / / distilldeep.ucd.ie / PeptideRanker / The isoelectric point (pI) and average hydrophilicity (GRAVY) were determined using the ProtParam tool in ExPASy. www.expasy.org / tools Toxicity prediction was performed using ToxinPred (…). https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php Allergenicity prediction was performed using AllerTOPv2.0 (). http: / / www.ddg-pharmfac.net / AllerTOP / ).

[0069] 3. Molecular docking:

[0070] Molecular docking was performed using AutoDock Vina software to connect the peptide to α-glucosidase (PDB ID: 2QMJ). The optimal docking conformation was selected based on the binding energy. The docking result file (pdbqt) was converted to a pdb file and then imported into Pymol software and the online website PLIP (…). https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index The results can be visualized and analyzed.

[0071] 4. Artificially synthesized polypeptides:

[0072] The screened peptides were artificially synthesized using the Fmoc-solid phase synthesis method, and the ability of each peptide to inhibit α-glucosidase was measured after synthesis.

[0073] 5. Determination of the type of inhibition of α-glucosidase:

[0074] Using PBS as a buffer system, and with a substrate PNPG concentration of 2.5 mmol / L, the enzyme reaction rates of different concentrations of peptide solutions (0, 2, 4, 6, and 8 mg / mL) and different enzyme concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 U / mL) were measured under different enzyme concentrations. Reaction curves were plotted with α-glucosidase concentration on the x-axis and reaction rate on the y-axis to determine the reversibility of the reaction. Using PBS as a buffer system, and with an enzyme concentration of 0.2 U / mL, the enzyme reaction rates of different concentrations of peptide solutions (0, 2, 4, 6, and 8 mg / mL) and different substrate concentrations (1, 2, 3, 4, and 5 mM) were measured under different substrate concentrations. Lineweaver-Burk curves were obtained by plotting the reciprocal of the substrate molar concentration (1 / [S]) on the x-axis and the reciprocal of the initial reaction rate (1 / V) on the y-axis. The type of inhibition of α-glucosidase by the peptide was determined based on the characteristics of the curves. Calculate V for competitive inhibition and non-competitive inhibition using equations (1) and (2) respectively. max and K m Then, the peptide concentration was plotted a second time using the slope and ordinate of the Lineweaver-Burk line, respectively, and the competitive inhibition constant K was calculated according to equations (3) and (4). ic Non-competitive inhibition constant K iu .

[0075] (1);

[0076] (2);

[0077] (3);

[0078] (4);

[0079] In the formula: V is the enzymatic reaction rate of α-glucosidase; K m K is the Michaelis constant; ic and K iu V represents the inhibition constants of the inhibitor-enzyme and the inhibitor-enzyme-substrate complex, respectively; max [I] represents the maximum reaction rate; [S] represents the peptide concentration; [I] represents the pNPG concentration.

[0080] 6. Effects of different pure peptides on postprandial blood glucose levels in mice:

[0081] The experimental design was parallel to Experiment 4.1. Mice were fed under standard laboratory conditions (25±1℃) and after 7 days of acclimatization, they were randomly divided into 10 groups for starch or maltose loading tests. The groups were as follows: (1) Starch control group (starch 1g / kg + physiological saline), (2) Starch positive control group (starch 1g / kg + acarbose 10mg / kg), (3) PGIGFPGPT group (starch 1g / kg + PGIGFPGPT 100mg / kg), (4) Maltose control group (maltose 2g / kg + physiological saline), (5) Maltose positive control group (maltose 2g / kg + acarbose 10mg / kg), (6) Maltose PGIGFPGPT group (maltose 2g / kg + PGIGFPGPT 100mg / kg). All mice were fasted for 12 h before the experiment, but were allowed free access to water. After fasting, the mice in the corresponding groups were gavaged with acarbose or pure peptide. 15 min later, the mice were gavaged with starch or maltose. Subsequently, blood samples were taken from the tail vein of mice at 0, 30, 60, 90, 120 and 180 min using a blood glucose meter, and blood glucose curves were plotted and the area under the curve was calculated.

[0082] result:

[0083] 1. The HAPH-1 fraction was identified by LC-MS / MS as containing 1527 peptides, with Mw < 3 kDa and peptide lengths ranging from 6 to 30 amino acids. Figure 7 Subsequently, the activity of bioactive peptides was predicted using the PeptideRanker website. Peptides with a score greater than 0.5 were considered to have potential bioactivity. To avoid false positives, peptides with scores greater than 0.8 (a total of 105) were further screened for prediction. Then, 41 non-toxic, non-allergenic peptides (P1-P41) with high potential bioactivity were identified using online analysis tools such as ExPASy, ToxinPred, and AllerTOP v2.0. The study showed that the structural characteristics of α-glucosidase inhibitory peptides include: 1) chain length 3–8; 2) N-terminus containing OH (Ser, Tyr, Thr) or basic amino acids (Lys, Arg, His); 3) C-terminus containing Ala or Met; 4) Pro is close to the C-terminus, preferably in the second-to-last position; 5) net charge is 0 or +1 at pH 7.0; 6) primarily based on hydrogen bonds and electrostatic interactions.

[0084] 2. The P39 (PGIGFPGPT) peptide was molecularly docked with α-glucosidase. The binding energies required for docking between the peptide and α-glucosidase are shown in Table 1. The degree of binding was evaluated by the binding energies of the ligand and acceptor; the smaller the binding energy, the easier it is for the two to bind and the stronger the interaction. The binding energy of P39 (PGIGFPGPT) with α-glucosidase is less than -9.0 kcal / mol. The interaction forces between P39 and α-glucosidase are shown in Table 2. P39 forms hydrogen bonds with the amino acid residues Asp 203, Arg 525, Ala 576, and Tyr 605 of α-glucosidase, and hydrophobic interactions with Tyr 299, Trp 406, Phe 575, and Gln 603. The binding of bioactive peptides to macromolecules mainly relies on various intermolecular forces, including hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic interactions, with hydrogen bonding playing a crucial role in stabilizing the docking complex. Therefore, it is hypothesized that P39 possesses strong α-glucosidase inhibitory activity.

[0085] Table 1: Binding energy required for peptide-α-glucosidase docking:

[0086] .

[0087] Table 2: Interaction forces between P39 and α-glucosidase:

[0088] .

[0089] 3. The peptide was confirmed as a novel peptide through the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) and manual search. Therefore, the peptide was synthesized in vitro to verify its α-glucosidase inhibitory activity. The purity of the synthesized peptides was >95%. Results are as follows: Figure 8 As shown, compared with HAPH-1, P39 exhibited significantly enhanced α-glucosidase inhibitory activity (p<0.05), IC50... 50 The value was 1.036 ± 0.017 mg / mL.

[0090] 4. Analyze the blood glucose levels of mice within 3 hours after gavage administration of P39 and starch or maltose. Figure 9The results showed that postprandial blood glucose levels in mice initially increased, reaching a maximum within 0.5 h. Blood glucose concentration decreased over time and returned to initial levels within 3 h. After gavage administration of starch, P39 inhibited the increase in postprandial blood glucose levels; compared with the control group, the acarbose and P39 groups significantly reduced the area under the blood glucose curve (p<0.05), consistent with the results of in vitro inhibition of α-glucosidase activity. The effect of the peptide on maltose-induced postprandial blood glucose levels in mice showed the same trend as the starch assay. Therefore, P39 has a significant hypoglycemic effect in vivo, suggesting that this peptide may have an inhibitory effect on α-glucosidase in vivo.

[0091] 5. Plot the enzyme concentration against the enzyme-catalyzed reaction rate. The results are as follows: Figure 10 As shown in Figure A, it can be observed that regardless of the presence or absence of the polypeptide, the reaction rate increases with increasing α-glucosidase concentration, and the trend lines for both the enzymatic reaction rate and α-glucosidase pass through the origin. As the polypeptide concentration gradually increases, the slope of the curve gradually decreases, indicating that the inhibitory effect of P39 on α-glucosidase is reversible. Lineweaver-Burk plots are used to study the inhibition type and kinetic parameters of the inhibitor. Furthermore, V... max Almost no change (Table 3). For example... Figure 10 As shown in Figure B, the fitted curves for P39 all intersect in the second quadrant, and V increases with increasing peptide concentration. max Decrease sequentially, K m The increase (Table 3) indicates that P39 inhibits α-glucosidase through a mixed-mode approach. As shown in Table 3, after linearly fitting the slope and ordinate of the straight line to the peptide mass concentration [I], the values ​​of the competitive inhibition constant Kic and the non-competitive inhibition constant Kiu were obtained according to the above formula. The K of P39... ic It is 4.54 mg / mL, K iu It was 7.51 mg / mL. K ic and K iu This reflects the inhibitory strength of the inhibitor; a smaller value indicates stronger inhibitory ability. P39 acts as a mixed inhibitor, where K... ic <K iu Therefore, this mixed inhibition is mainly competitive inhibition, meaning that the inhibitor binds to the enzyme more readily than the enzyme-substrate complex. This result is consistent with the IC50 findings mentioned above. 50 The conclusions are consistent.

[0092] Table 3: Inhibition Types:

[0093] .

[0094] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the present invention. Those skilled in the art should understand that these are merely illustrative examples, and all modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A hybrid inhibitory α-glucosidase activity-inhibiting hippocampal peptide, characterized in that: Including polypeptides with amino acid sequences as shown in SEQ ID NO.

1.

2. The method for preparing the mixed-type α-glucosidase-inhibiting hippocampal peptide according to claim 1, characterized in that: It is artificially synthesized.

3. The use of the mixed-type α-glucosidase-inhibiting hippocampal peptide of claim 1 in the preparation of hypoglycemic drugs.

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