A hypoglycemic polypeptide dpgw from yangjiang douchi and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a hypoglycemic polypeptide DPDGW from Yangjiang douchi and application thereof, the hypoglycemic polypeptide is mainly DPDGW, or is any one or more of LPWPH, FPAYGH, DGWFR, YDFR, NWEFR or SFFPR. The present application first separates and identifies the hypoglycemic polypeptide with a clear sequence from Yangjiang douchi. In particular, the polypeptide DPDGW has a very low half-inhibitory concentration of DPP-IV, and the activity is the most outstanding. The polypeptide is derived from a traditional fermented food, has high safety, and can be widely applied to preparation of hypoglycemic drugs, health foods or dietary supplements and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a hypoglycemic polypeptide DPDGW derived from Yangjiang fermented soybeans and its applications. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, with type 2 diabetes mellitus (T2DM) accounting for over 90% of all diabetes cases. The pathogenesis of T2DM primarily involves insulin resistance and impaired pancreatic β-cell function, leading to an imbalance in glycemic regulation. Long-term hyperglycemia can cause various complications such as cardiovascular disease, nephropathy, and retinopathy, seriously threatening patients' lives and health. Currently, commonly used hypoglycemic drugs in clinical practice include metformin, sulfonylureas, alpha-glucosidase inhibitors, and DPP-IV inhibitors. However, long-term use of these chemically synthesized drugs is prone to side effects, such as gastrointestinal discomfort, hypoglycemia risk, and liver and kidney damage. Therefore, finding safe and effective hypoglycemic active ingredients from natural food resources has become a current research hotspot.

[0003] Currently, research on natural multi-target hypoglycemic peptides mainly focuses on raw materials such as soybeans and rice, while the bioactive peptide resources of fermented foods like fermented black soybeans (douchi) have not been fully explored. Douchi, a traditional Chinese fermented soybean product, has a long history and holds an important place in Asian culinary culture. Douchi can be classified into mold-based douchi (such as Yangjiang douchi) and bacterial-based douchi (such as natto) according to the type of fermenting microorganisms. During fermentation, the proteases produced by the microorganisms hydrolyze soybean protein into various bioactive peptides, endowing douchi with multiple physiological functions. Existing studies have shown that douchi extracts possess various biological activities such as antioxidant, blood pressure-lowering, and blood lipid-lowering effects. For example, Iwasaki Eriko found in her research that bacterial douchi extract has a preventive and therapeutic effect on diabetes, and its mechanism is related to improving insulin resistance and regulating glucose and lipid metabolism. Patent CN118662545A discloses a composition containing Tremella fuciformis polysaccharide and fermented soybean extract, which has hypoglycemic effects. However, its active ingredient is a complex of polysaccharide and crude fermented soybean extract, without identifying the specific peptide sequence or verifying its function. Patent CN118620976A discloses a fermented soybean polypeptide extract and its preparation method and application. This invention prepares fermented soybean polypeptides via enzymatic hydrolysis and verifies its in vitro DPP-IV inhibitory activity. However, it uses Bacillus subtilis-fermented fermented soybean as raw material and does not isolate and identify the specific active peptide sequence, making it impossible to clarify the molecular basis of its hypoglycemic activity. Patent CN119385302A discloses a method for preparing a composition containing light fermented soybean extract for controlling blood sugar in type II diabetes patients. This invention uses light fermented soybean as raw material to prepare the extract and combines it with other components, but its core active ingredient is still the crude extract, without delving into the mechanism at the polypeptide sequence level.

[0004] In the study of glucose-lowering peptide targets, dipeptidyl peptidase IV (DPP-IV) is an important target for the treatment of type 2 diabetes mellitus (T2DM). DPP-IV can rapidly degrade incretin GLP-1 and GIP, leading to decreased insulin secretion and elevated blood glucose. Therefore, inhibiting DPP-IV activity can effectively prolong the duration of action of endogenous GLP-1, promote insulin secretion, and lower blood glucose levels. Furthermore, α-glucosidase and α-amylase are key enzymes in carbohydrate digestion and metabolism; inhibiting their activity can delay glucose release and absorption, thereby controlling postprandial hyperglycemia. Currently, screening for glucose-lowering peptides with multi-target inhibitory activity from food-derived proteins has become a research frontier. However, there are no reports of isolating a single polypeptide component with a defined amino acid sequence and multi-target glucose-lowering activity from Yangjiang fermented black soybeans.

[0005] Yangjiang fermented black soybeans (douchi) are a traditional specialty fermented food from Yangjiang City, Guangdong Province. Made from black soybeans through natural fermentation with Aspergillus, its unique fermentation process and microbial system may produce bioactive peptide profiles different from other douchi. However, current research on hypoglycemic active peptides from Yangjiang douchi is lacking, with insufficient systematic isolation and identification of these peptides and a lack of in-depth elucidation of their hypoglycemic mechanisms. Therefore, isolating and identifying hypoglycemic peptide sequences from Yangjiang douchi and clarifying their hypoglycemic mechanisms is of significant theoretical and practical value for developing novel natural hypoglycemic functional factors and promoting the high-value utilization of the Yangjiang douchi industry. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a hypoglycemic polypeptide DPDGW derived from Yangjiang fermented soybeans and its application. This polypeptide has advantages such as strong hypoglycemic activity and good gastrointestinal stability, providing a new option for developing novel natural hypoglycemic functional factors.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0008] Firstly, this invention provides a hypoglycemic polypeptide DPDGW (SEQ ID NO:1) derived from Yangjiang fermented black soybeans, with the amino acid sequence Asp-Pro-Asp-Gly-Trp. This polypeptide, derived from the traditional fermented food Yangjiang fermented black soybeans, exhibits the most prominent DPP-IV inhibitory activity in in vitro DPP-IV evaluation, with an IC50 value of [missing value]. 50 With a concentration of only 83.25 μM and excellent resistance to gastrointestinal digestion, it is the optimal target hypoglycemic peptide.

[0009] Furthermore, the present invention also provides a hypoglycemic polypeptide derived from Yangjiang fermented soybean, any one or more of LPWPH (SEQ ID NO:2), FPAYGH (SEQ ID NO:3), DGWFR (SEQ ID NO:4), YDFR (SEQ ID NO:5), NWEFR (SEQ ID NO:6) or SFFPR (SEQ ID NO:7); wherein the sequence of LPWPH is Leu-Pro-Trp-Pro-His, the sequence of FPAYGH is Phe-Pro-Ala-Tyr-Gly-His, the sequence of DGWFR is Asp-Gly-Trp-Phe-Arg, the sequence of YDFR is Tyr-Asp-Phe-Arg, the sequence of NWEFR is Asn-Trp-Glu-Phe-Arg, and the sequence of SFFPR is Ser-Phe-Phe-Pro-Arg. These peptides also originate from Yangjiang fermented black soybeans. They are food-derived natural active peptides with small molecular weights. They not only have significant hypoglycemic activity, but also have good stability in the gastrointestinal environment, are easily absorbed, and have high safety.

[0010] Furthermore, the present invention also provides a nucleic acid molecule encoding the aforementioned hypoglycemic polypeptide. Considering the degeneracy of codons, any nucleic acid sequence capable of translating the aforementioned amino acid sequence is within the scope of protection of the present invention.

[0011] Furthermore, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule. The expression vector may be a plasmid, granulocyte, bacteriophage, or viral vector, etc. Those skilled in the art can operatively link the nucleic acid molecule to a suitable promoter and downstream of a regulatory sequence, depending on the selection of the actual host cell, to achieve the expression of the polypeptide.

[0012] Furthermore, the present invention provides a host cell comprising the above-described expression vector. The host cell can be a prokaryotic cell (such as *Escherichia coli*) or a eukaryotic cell (such as yeast or mammalian cells). By introducing the expression vector into the host cell, recombinant expression and large-scale preparation of the hypoglycemic peptide can be achieved.

[0013] Secondly, this invention provides the application of the above-mentioned hypoglycemic peptides in the preparation of hypoglycemic products. These hypoglycemic products include, but are not limited to, hypoglycemic drugs, hypoglycemic health foods, hypoglycemic functional foods, or hypoglycemic dietary supplements. Because these peptides are derived from Yangjiang fermented black soybeans, they have extremely high food safety, making them particularly suitable for long-term use in health foods and dietary supplements.

[0014] Thirdly, this invention provides the application of the above-mentioned hypoglycemic peptides in the preparation of target inhibitors or modulators. Specifically, it includes:

[0015] (1) Application of the hypoglycemic peptide in the preparation of DPP-IV inhibitors. This type of peptide can target and inhibit DPP-IV enzyme activity, reduce the degradation of incretins (such as GLP-1), and thus maintain blood glucose homeostasis;

[0016] (2) The application of the hypoglycemic peptide in the preparation of α-glucosidase and / or α-amylase inhibitors. This type of peptide can target and inhibit the activity of digestive enzymes of carbohydrates in the intestine, delay the release and absorption of glucose, and effectively control the rise in postprandial blood glucose;

[0017] (3) The application of the glucose-lowering peptides in the preparation of PI3K / AKT signaling pathway activators, FOXO1 transcription inhibitors, PEPCK and G6PC expression downregulators, and / or glycogen synthase expression upregulators. These peptides can activate the PI3K / AKT signaling pathway, inhibit the transcriptional activity of FOXO1, thereby downregulating the expression of key gluconeogenesis enzymes PEPCK and G6PC, while upregulating the expression of glycogen synthase, ultimately reducing hepatic glucose output, increasing hepatic glycogen storage, and improving hepatocyte insulin resistance.

[0018] Fourthly, the present invention provides a hypoglycemic composition comprising an effective amount of the aforementioned hypoglycemic peptide and a pharmaceutically or food-grade acceptable carrier or excipient. The carrier or excipient includes, but is not limited to, fillers, disintegrants, lubricants, binders, preservatives, antioxidants, flavoring agents, or solvents, to meet the formulation requirements of different products.

[0019] Furthermore, the dosage form of the hypoglycemic composition is capsules, tablets, granules, powders, or oral liquids. Considering the characteristics of polypeptide drugs and foods, the above dosage forms can all be prepared using conventional pharmaceutical processes in the art, which not only facilitates carrying and administration but also effectively protects polypeptide activity and improves bioavailability.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] Firstly, this invention is the first to isolate and identify hypoglycemic peptides (DPDGW, LPWPH, FPAYGH, DGWFR, YDFR, NWEFR, SFFPR) with distinct amino acid sequences from Yangjiang fermented black beans, filling a gap in the research on specific hypoglycemic peptides from Yangjiang fermented black beans. These peptides are derived from traditional fermented foods, have extremely high safety for consumption, and have small molecular weights, making them easily absorbed and utilized by the body.

[0022] Secondly, these hypoglycemic peptides exhibit excellent gastrointestinal stability and multi-target hypoglycemic activity. After simulated gastrointestinal digestion, their hypoglycemic activity not only did not decrease but was significantly enhanced, effectively overcoming the problem of easy degradation and inactivation of conventional food-derived peptides after oral administration. They can simultaneously target and inhibit the activity of DPP-IV, α-glucosidase, and α-amylase, synergistically lowering blood glucose through multiple pathways such as delaying carbohydrate absorption and promoting insulin secretion, demonstrating extremely strong multi-target synergistic hypoglycemic potential.

[0023] Finally, this invention elucidates the molecular mechanism by which these peptides improve insulin resistance in hepatocytes. They effectively reduce hepatic glucose output and increase hepatic glycogen storage by activating the PI3K / AKT signaling pathway, inhibiting FOXO1 transcription, downregulating key gluconeogenesis enzymes, and upregulating glycogen synthase. In particular, the peptide DPDGW binds tightly to the active site of DPP-IV, exhibiting an extremely low half-maximal inhibitory concentration (WMC) for DPP-IV, demonstrating the strongest binding specificity and inhibitory efficacy at the molecular level. This provides solid theoretical support and a novel material basis for the development of new natural hypoglycemic functional factors. Attached Figure Description

[0024] Figure 1 The graph shows the in vitro hypoglycemic activity evaluation results of DPF ultrafiltration fractions; where (A) is the inhibition rate against α-glucosidase, (B) is the inhibition rate against α-amylase, (C) is the inhibition rate against DPP-IV, and (D) is the IC50 inhibition rate against α-glucosidase. 50 Value (E) is the IC50 value for α-amylase inhibition. 50 Value (F) is the DPP-IV inhibitor IC. 50 value.

[0025] Figure 2 The figure shows the effect of in vitro simulated gastrointestinal digestion on hypoglycemic activity; where (A) is the inhibition rate of DPE-4 on different enzymes, and (B) is the half-inhibition rate (IC50) of DPU-4 and DPE-4. 50 Value comparison.

[0026] Figure 3 This is a schematic diagram of the screening process for hypoglycemic peptide components.

[0027] Figure 4 The diagram shows the docking results of the synthetic peptide with the DPP-IV target protein molecule; where (A) is FPAYGH, (B) is LPWPH, (C) is DPDGW, (D) is DGWFR, (E) is YDFR, (F) is NWEFR, and (G) is SFFPR. Detailed Implementation

[0028] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings.

[0029] Example 1

[0030] The purpose of this embodiment is to provide a water extract (DPF) of fermented soybean polypeptide and its preparation method, as follows:

[0031] Yangjiang fermented black beans (purchased from Guangdong Yangfan Food Co., Ltd., product number 6972031245658) were collected by peeling off the outer skin and collecting the fermented black bean flesh. Deionized water was added at a material-to-liquid ratio of 1:4 (w / w), and the mixture was ground into a homogenate on ice. To avoid damaging proteins and peptides with high temperatures and to improve extraction efficiency, extraction was carried out at 4℃ with low-speed stirring at 80 rpm for 1 hour. The homogenate was then centrifuged at 4℃ and 20,000 × g for 30 minutes. The supernatant was collected and dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 500 Da to remove small molecule impurities such as salt and free amino acids. The retentate was freeze-dried to obtain the fermented black bean polypeptide aqueous extract (DPF). The above preparation process was repeated three times.

[0032] The dry weight yield of the soybean polypeptide aqueous extract (DPF) prepared by the above method was determined to be 30.5%±1.5%, the protein content was 56.8%±1.4%, the polypeptide content was 65.0%±2.1%, the isoflavone content was 4.7%±0.3%, and the polysaccharide content was 3.2%±0.2%, of which the polypeptides with a molecular weight <3kDa accounted for approximately 93%.

[0033] Meanwhile, the DPF prepared in this embodiment was evaluated for its in vitro hypoglycemic activity by measuring its inhibitory activity against α-amylase, α-glucosidase, and DPP-IV. The results showed that the inhibitory effects of DPF on all three enzymes were significantly dose-dependent, with DPF exhibiting the best inhibitory effect on α-amylase (IC50). 50 =15.36±0.72mg / mL), followed by DPP-IV inhibition (IC50). 50 =24.11±1.17 mg / mL) and α-glucosidase inhibition effect (IC50) 50 =24.27±1.23 mg / mL). This result indicates that DPF has good potential for multi-target hypoglycemic action in vitro and is an excellent source for screening natural hypoglycemic peptides.

[0034] Based on the aforementioned research, a DPF dispersion with a mass concentration of 30 mg / mL was accurately prepared. Separation and fractionation were performed using 10 kDa, 5 kDa, and 3 kDa ultrafiltration membranes to obtain four fractions with molecular weights >10 kDa, 10-5 kDa, 5-3 kDa, and <3 kDa, named DPU-1, DPU-2, DPU-3, and DPU-4, respectively. Following the aforementioned method, the in vitro hypoglycemic activity of each fraction was evaluated. The results showed that fraction DPU-4 (molecular weight <3 kDa) exhibited the strongest inhibitory activity against the three enzymes; therefore, DPU-4 was selected for further research.

[0035] Example 2

[0036] Based on Example 1 above, this example further uses the fermented soybean polypeptide aqueous extract DPF prepared in Example 1 as the research object to screen for hypoglycemic active peptide components with good stability, as detailed below:

[0037] 2.1 Ultrafiltration purification of DPF and its in vitro hypoglycemic activity investigation

[0038] A DPF dispersion with a mass concentration of 30 mg / mL was accurately prepared. Separation and fractionation were performed using 10 kDa, 5 kDa, and 3 kDa ultrafiltration membranes to obtain hydrolysis products with different molecular weights, resulting in four fractions: >10 kDa, 10-5 kDa, 5-3 kDa, and <3 kDa. The permeate and retentate were collected and named DPU-1, DPU-2, DPU-3, and DPU-4, respectively. These were freeze-dried to prepare peptide powder, and their enzyme inhibitory activity was determined according to the aforementioned method. The experimental results are as follows: Figure 1 As shown.

[0039] The experimental results show that the inhibitory effects of the four components DPU-1 to DPU-4 on α-glucosidase, α-amylase, and DPP-IV are dose-dependent, and the inhibitory activity is stronger with increasing concentration. Among them, the component DPU-4 with the smallest average molecular weight showed the strongest activity and will be used for subsequent research.

[0040] 2.2 In vitro simulation of gastrointestinal digestion

[0041] The DPU-4 was subjected to in vitro simulated gastrointestinal digestion, as follows:

[0042] Prepare a sample solution with a final concentration of 100 mg / mL, and hydrolyze it sequentially with artificial gastric juice (pepsin) and artificial intestinal juice (trypsin), as follows:

[0043] The sample solution was preheated at 37℃ for 10 min, then pepsin / peptide ratio of 1:10 (w / w) was added, pH was adjusted to 2.0, and enzymatic hydrolysis was performed in a water bath at 37℃ for 3 h. The pH was adjusted to 7.5, and trypsin was added to the reaction system at a trypsin / peptide ratio of 1:10 (w / w), and the reaction was continued at 37℃ for 3 h. After hydrolysis, the reaction system was heated in boiling water for 10 min to inactivate the trypsin. After cooling, the sample was centrifuged at 10000×g for 20 min, the supernatant was collected, freeze-dried, and stored at -20℃ for later use. This yielded a sample that had undergone in vitro simulated gastrointestinal digestion and was recorded as DPE-4.

[0044] The in vitro hypoglycemic activity of DPE-4 was tested, and the hypoglycemic activities of DPE-4 and DPU-4 were compared. The experimental results are attached. Figure 2 As shown.

[0045] like Figure 2 As shown in Figure A, the digestive product DPE-4, after being digested in a simulated gastrointestinal tract, still exhibits increased in vitro hypoglycemic activity with increasing dose concentration. Figure 2 As shown in Figure B, compared with the DPU-4 component, the simulated gastrointestinal digestive product DPE-4 significantly enhanced the inhibitory activities of α-glucosidase, α-amylase, and DPP-IV (P<0.05).

[0046] IC50 of the simulated digestion product DPE-4 50 The values, from smallest to largest, are: DPP-IV (IC 50 =5.56±0.96mg / mL) < α-amylase (IC50) 50 =6.8±1.69mg / mL) < α-glucosidase (IC50) 50 =12.55±1.12mg / mL), indicating that DPE-4 exhibited the best in vitro inhibitory activity against DPP-IV.

[0047] The above experimental results may be related to the fact that enzymatic hydrolysis of macromolecules releases more and smaller bioactive peptides that occupy the active sites of DPP-IV. Natural macromolecular proteins, due to their dense spatial structure, have active sites encapsulated within the molecule, making them difficult to bind to enzymes and exert inhibitory effects. However, after in vitro simulated gastrointestinal digestion, under the action of pepsin and trypsin, the peptide bonds of proteins are specifically cleaved, especially involving changes in structural forms such as α-helices, β-turns, and random coils. This leads to the exposure and release of functional peptide fragments, thereby significantly enhancing the in vitro hypoglycemic ability of DPE-4. Considering that the DPE-4 component has the strongest in vitro hypoglycemic activity and a better inhibitory effect on DPP-IV, DPP-IV was used as a classic in vitro hypoglycemic target for computer-simulated screening to narrow down the screening range of hypoglycemic peptides from Yangjiang fermented soybeans.

[0048] 2.3 Identification and Screening of Glycemic Peptide Sequences in DPE-4

[0049] Peptide analysis of the DPE-4 fraction was performed by LC-MS / MS. A total of 24,595 peptide sequences were identified in the DPE-4 fraction, of which 303 had an ALC ≥ 98%. The experimental results are shown in Table 1.

[0050] Table 1. Peptides in DPE-4 identified by de novo sequencing.

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] The PeptideRanker tool can be used to predict the potential biological activity of peptides. Within the score range of 0-1, peptide sequences with a score > 0.8 exhibit significant hypoglycemic activity. (Screenshot of the screening flowchart follows.) Figure 3 As shown in Table 2, 25 peptides with hypoglycemic potential were identified from the DPE-4 fraction after computer-based hierarchical screening.

[0057] Table 2 Results of peptide sequence screening in DPE-4

[0058] The toxicity, sensitization, hydrophobicity, water solubility, isoelectric point, and net charge of these 25 peptide sequences were comprehensively analyzed, and the binding ability of the peptides to the DPP-IV target protein was further evaluated.

[0059] The results showed that these peptides were all between 4 and 6 amino acids in length, with molecular weights ranging from 406.18 to 750.34 Da, isoelectric points from 3 to 11.11, net charges from -2 to +1, and hydrophobic indices from 5 to 14.38. All peptide sequences were non-toxic and non-sensitizing. Based on previous studies confirming that short peptide sequences of 2 to 8 amino acids are effective DPP-IV inhibitory peptides, this is consistent with our screening results. Hydrophobicity plays a crucial role in the inhibitory activity of hypoglycemic peptides; our peptide sequences exhibited 100% hydrophobic properties, suggesting that the strong hypoglycemic effect of DPE-4 may be related to its strong hydrophobic characteristics.

[0060] The binding energies of the seven peptides FPAYGH (SEQ ID NO:3), LPWPH (SEQ ID NO:2), DPDGW (SEQ ID NO:1), DGWFR (SEQ ID NO:4), YDFR (SEQ ID NO:5), NWEFR (SEQ ID NO:6), and SFFPR (SEQ ID NO:7) are -10.6 kcal / mol, -10.3 kcal / mol, -9.4 kcal / mol, -9.4 kcal / mol, -9.3 kcal / mol, -9.2 kcal / mol, and -8.8 kcal / mol, respectively. Their extremely low binding energies indicate that these seven peptide sequences may have the potential to strongly bind to the DPP-IV active site and can be further studied. The amino acid sequences of the above candidate hypoglycemic peptides are shown in Table 3.

[0061] Table 3. Amino acid sequences of 7 candidate hypoglycemic peptides

[0062] Example 3

[0063] The purpose of this embodiment is to evaluate the activity of candidate hypoglycemic peptides. The experimental method is as follows:

[0064] Seven candidate hypoglycemic peptides—FPAYGH, LPWPH, DPDGW, DGWFR, YDFR, NWEFR, and SFFPR—obtained through computer-based virtual screening were synthesized in a solid-phase manner (by Sangon Biotech (Shanghai) Co., Ltd., with a purity ≥95%), and the following analyses were performed:

[0065] 3.1 Study on the molecular docking mechanism of blood sugar-lowering peptides from fermented soybeans

[0066] Inhibition of DPP-IV activity is one of the core mechanisms by which the hypoglycemic peptide from fermented soybean exerts its in vitro hypoglycemic effect. Its catalytic activity depends on three main binding pockets at the active site: the hydrophobic S1 pocket (containing Tyr547, Ser630, Tyr631, Val656, Trp659, Tyr662, Tyr666, Val711, and His740 residues), the charged S2 pocket (including Arg125, Glu205, Glu206, Ser209, Phe357, Arg358, Tyr662, and Asn710), and the inhibitor-related S3 pocket (composed of Phe357, Arg358, and Tyr547). Figure 4 As shown, the seven soybean blood sugar-lowering peptides screened in this study—FPAYGH, LPWPH, DPDGW, DGWFR, YDFR, NWEFR, and SFFPR—can all bind tightly to the active site of DPP-IV. The specific docking portion of each peptide is as follows:

[0067] like Figure 4 As shown in Figure A, FPAYGH forms hydrogen bonds with Lys122, Arg125, Ser630, His740, Asn710, Asp709, Asp545, Glu205, and Glu206 residues in the active pocket of DPP-IV; it also forms C-H bonds with Arg125, Ser630, and Glu205, constructing a stable hydrogen bond network; and it forms π-π stacking interactions with Phe357 and Trp629, and π-alkyl interactions with Tyr662 and Tyr666, further enhancing the stability of peptide-enzyme binding. The binding of nine S2, S1, and S3 pocket residues, including Arg125 and Ser630, directly hinders the binding of the substrate to the active site of DPP-IV, revealing the reason for the extremely low binding energy of FPAYGH.

[0068] like Figure 4 As shown in Figure B, LPWPH forms hydrogen bonds with Lys122, Arg125, Asp739, Asn710, Asp709, and Glu205 of DPP-IV, and carbon-hydrogen bonds with Asp709. It also forms π-σ interactions with Tyr662 and Tyr666 in the S1 pocket, π-π stacking interactions with Tpr629, and π-alkyl interactions with Val656 and Tyr547 (in the S1 and S3 pockets). The synergistic effect of these multiple types of hydrophobic interactions reveals the main reason for the binding affinity between LPWPH and DPP-IV.

[0069] like Figure 4 As shown in Figure C, DPDGW forms hydrogen bonds with Arg125, Lys554, Trp629, Tyr547, and Asn710 of DPP-IV, and carbon-hydrogen bonds with Glu206. It also forms π-π stacking interactions with Trp629 and π-alkyl interactions with Phe357 and Tyr666. The DPDGW binding sites completely cover the three core pockets, comprehensively preventing substrate entry into the active site, further demonstrating its potent in vitro DPP-IV inhibitory activity.

[0070] like Figure 4 As shown in Figure D, DGWFR forms an attractive charge interaction with Glu206 of DPP-IV. It also forms hydrogen bonds with Arg125, Ser630, Tyr631, Tyr662, Asn710, Tyr547, Glu205, and Glu206; a carbon-hydrogen bond with Asn710; and a π-donor hydrogen bond with Phe357, collectively constructing a stable hydrogen bond network. Simultaneously, it forms π-π stacking interactions with Tyr547, Trp629, Trp629, and Trp629, and a π-alkyl interaction with Lys554. This synergistic effect of multiple interactions indicates that the well-known characteristic of DGWFR lacking the N-terminal second proline residue allows it to form a stable complex with DPP-IV, significantly enhancing its binding affinity.

[0071] like Figure 4 As shown in Figure E, YDFR forms charge-attracting interactions with Glu205, Asp709, and Asp739 of DPP-IV, enhancing its electrostatic binding to the active pocket of DPP-IV. It also forms hydrogen bonds with Arg125, Arg358, Ser630, Tyr631, His740, Glu206, Asp709, and Glu205, and a π-donor hydrogen bond with Tyr666. Furthermore, it forms π-π stacking interactions with Phe357 and Tyr547. This indicates that YDFR balances electrostatic and hydrophobic interactions, ensuring its stable occupancy of the active site.

[0072] like Figure 4 As shown in Figure F, NWEFR forms a charge-attractive interaction with Asp545 of DPP-IV; hydrogen bonds with Arg125, Gln553, Lys554, Ser630, Glu206, Val546, and Asp545; carbon-hydrogen bonds with Ser552; and π-π stacking interactions with Phe357 and Trp629. This indicates that NWEFR achieves stable binding with DPP-IV through a synergistic effect of electrostatics, hydrogen bonding, and hydrophobic interactions.

[0073] like Figure 4 As shown in G, SFFPR forms a charge-attracting interaction with Glu205 of DPP-IV and a salt bridge with Glu206, significantly enhancing its electrostatic binding ability. Simultaneously, it forms hydrogen bonds with eight S1, S2, and S3 pocket residues (Arg125, Ser209, Ser630, Tyr547, Asn710, Glu206, Tyr662, and GLU205), π-π stacking interactions with Tyr547 and Trp629, and π-alkyl interactions with Lys554. The synergy of the salt bridge, hydrogen bond network, and hydrophobic interactions further enhances the binding stability and specificity of SFFPR.

[0074] Comprehensive analysis revealed that all seven soybean-derived hypoglycemic peptides formed stable interactions with the active site of DPP-IV, exhibiting a high degree of consistency in their binding modes. These interactions were achieved through a synergistic effect of hydrogen bond networks, electrostatic interactions, and hydrophobic interactions, resulting in highly efficient binding to DPP-IV. Among these, the hydrogen bond network (including conventional hydrogen bonds, C-H bonds, and π-donor hydrogen bonds) is the primary determinant of peptide-enzyme interactions, directly determining the specificity and stability of the binding between soybean-derived hypoglycemic peptides and DPP-IV. Furthermore, the abundance of hydrogen bond networks is positively correlated with the inhibitory efficacy of DPP-IV. Charge attraction and salt bridges, acting as strong electrostatic interactions, not only provide powerful forces for peptide-enzyme binding but also guide the peptides into the active pocket of DPP-IV in the correct conformation, which is crucial for maintaining the stable conformation of the peptide-enzyme complex. π-related interactions (including π-π stacking, π-alkyl, and π-σ interactions) mainly depend on aromatic amino acids (such as phenylalanine, tyrosine, and tryptophan) in the peptide. They play a key role in the hydrophobic environment of the DPP-IV active pocket, effectively excluding water molecules and greatly enhancing peptide-enzyme binding affinity.

[0075] The seven hypoglycemic peptides screened in this study all stably occupied the S1, S2, and S3 core active pockets of DPP-IV. Through the synergistic effect of various intermolecular interactions, they effectively inhibited the binding of substrates to the active sites of DPP-IV. This result corroborates the previous in vitro DPP-IV inhibitory activity experiments, clearly revealing the molecular mechanism by which Yangjiang fermented soybean hypoglycemic peptides exert their DPP-IV inhibitory effect, providing important molecular-level support for subsequent pure peptide activity verification and application development. Furthermore, combined with previous studies on α-amylase and α-glucosidase inhibitory activities, this further confirms the multi-target synergistic hypoglycemic potential of fermented soybean hypoglycemic peptides, providing a scientific basis for their development as a natural hypoglycemic functional component.

[0076] 3.2 Evaluation of DPP-IV inhibitory activity

[0077] Following the method of Hong et al., different concentrations of DPF sample solutions, Gly-Pro-pNA (1.6 mM), and DPP-IV (10 U / L) were prepared using Tris-HCl buffer (0.1 M, pH 8.0). 25 μL of Gly-Pro-pNA substrate and 25 μL of sample were incubated in a 96-well plate at 37 °C for 10 min. Subsequently, 50 μL of DPP-IV was added, and the mixture was incubated at 37 °C for 60 min. Sitagliptin phosphate was used as a positive control, and absorbance was measured at 405 nm, repeated three times, with the average value taken.

[0078] The formula for calculating the DPP-IV inhibition rate is as follows:

[0079] DPP-IV Inhibition (%) = [1 - (A) x -A xo ) / (B xo -B o ]×100;

[0080] Among them, A x A represents the absorbance value of the sample group. xo B represents the absorbance value of the sample background group. xo B is the absorbance value of the control group; o The absorbance values ​​are for the blank control group.

[0081] After determining the inhibition rate of each synthetic peptide at different concentrations, nonlinear regression fitting was performed using GraphPad Prism software to calculate the half-maximal inhibitory concentration (IC50) of each peptide, expressed as mass concentration. 50 (mg / mL)]; further, based on the relative molecular mass of each peptide, it is converted into the half-maximal inhibitory concentration (IC50) expressed as molar concentration [IC50]. 50 [(μM)], the experimental results are shown in Table 4.

[0082] The experimental results are shown in Table 4:

[0083] Table 4. DPP-IV inhibitory activity of synthetic peptides.

[0084]

[0085] The above experimental results show that the four peptides FPAYGH, LPWPH, DPDGW, and DGWFR exhibit significant DPP-IV inhibitory effects, with DPDGW showing the most prominent hypoglycemic activity (IC50). 50 The value reached 83.25 μM. At the same time, DPDGW also has good anti-gastrointestinal digestive stability. Compared with other peptides, it shows better potential for adjuvant treatment of T2DM and has become the optimal target hypoglycemic peptide screened in this invention, laying the foundation for subsequent verification of the hypoglycemic mechanism at the cellular level and the development of functional foods.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A hypoglycemic polypeptide DPDGW derived from Yangjiang douchi, characterized in that, It is DPDGW, with the amino acid sequence Asp-Pro-Asp-Gly-Trp, as shown in SEQ ID NO:

1.

2. A hypoglycemic polypeptide derived from Yangjiang douchi, characterized in that, Selected from any one or more of the sequences shown in SEQ ID NO:2 to SEQ ID NO:7; wherein, the sequence of SEQ ID NO:2 is Leu-Pro-Trp-Pro-His (LPWPH), the sequence of SEQ ID NO:3 is Phe-Pro-Ala-Tyr-Gly-His (FPAYGH), the sequence of SEQ ID NO:4 is Asp-Gly-Trp-Phe-Arg (DGWFR), the sequence of SEQ ID NO:5 is Tyr-Asp-Phe-Arg (YDFR), the sequence of SEQ ID NO:6 is Asn-Trp-Glu-Phe-Arg (NWEFR), and the sequence of SEQ ID NO:7 is Ser-Phe-Phe-Pro-Arg (SFFPR).

3. A nucleic acid molecule encoding the hypoglycemic polypeptide of claim 1 or 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. The use of the hypoglycemic peptide according to claim 1 or 2 in the preparation of hypoglycemic products.

7. The application according to claim 6, characterized in that, The blood sugar lowering products include blood sugar lowering drugs, blood sugar lowering health foods, blood sugar lowering functional foods, or blood sugar lowering dietary supplements.

8. The use of the hypoglycemic peptide of claim 1 or 2 in the preparation of DPP-IV inhibitors, and / or in the preparation of α-glucosidase and / or α-amylase inhibitors; and / or in the preparation of PI3K / AKT signaling pathway activators, FOXO1 transcription inhibitors, PEPCK and G6PC expression downregulators, glycogen synthase expression upregulators, and / or products that improve hepatocyte insulin resistance.

9. A hypoglycemic composition, characterized in that, It contains an effective amount of the hypoglycemic peptide of claim 1 or 2, as well as a pharmaceutically or food-grade acceptable carrier or excipient.

10. The hypoglycemic composition according to claim 9, characterized in that, The composition is available in the form of capsules, tablets, granules, powders, or oral liquids.

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