A dual-target active peptide derived from scallop skirt, screening method and application thereof

Abstract

The application discloses a double-target active peptide derived from scallop skirt, and a screening method and application thereof, characterized in that the amino acid sequence of the active peptide is selected from at least one of VDMFWR, PAGGR and SSCLR. Based on a molecular simulation technology, the three novel active peptides are efficiently obtained from scallop skirt enzymatic hydrolysate through virtual enzymolysis, ultrafiltration grading, peptidomics identification, multi-level computer virtual screening and molecular docking verification. The active peptide has the advantages of significant angiotensin converting enzyme and alpha-amylase double-inhibitory activity, and can play the functions of reducing blood pressure and blood sugar through multiple pathways such as inhibiting ACE / alpha-amylase, regulating NO / ET-1 balance, resisting oxidation, improving glycolipid metabolism and the like at a cell level.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method derived from scallops ( Argopecten irradians The study focuses on dual-target active peptides with angiotensin-converting enzyme (ACE) inhibitory activity and α-amylase inhibitory activity, along with their screening methods and applications. Background Technology

[0002] Hypertension and hyperglycemia are two major chronic health problems affecting approximately one-third of the world's adult population. They often coexist and significantly increase the risk of cardiovascular and cerebrovascular diseases. Currently, commonly used synthetic antihypertensive drugs (such as captopril) and hypoglycemic drugs (such as metformin and acarbose) can effectively control related indicators, but long-term use can easily cause side effects such as dry cough, gastrointestinal discomfort, hypoglycemia, and drug tolerance. Therefore, developing functional active ingredients with high safety and few side effects from natural sources is of significant practical importance.

[0003] With the development of bioinformatics and computational biology, molecular simulation technology has become an important auxiliary tool for screening bioactive peptides. By performing virtual enzymatic digestion, activity prediction, and molecular docking on target proteins in protein databases, potentially highly active sequences can be quickly identified before large-scale peptide synthesis and experimentation, effectively overcoming the shortcomings of the traditional "enzymatic digestion-separation-activity tracking" path, which is labor-intensive and time-consuming.

[0004] Scallops are an important farmed shellfish in my country, with a huge annual output. During processing, the scallop skirt, a major byproduct, accounts for approximately 27.8% of the whole scallop's weight and is currently mostly processed into low-value products such as dried goods or seasonings. Studies have shown that scallop skirts are rich in crude protein (up to 68% or more) and have a balanced amino acid composition, making them an ideal raw material for preparing bioactive peptides. In recent years, studies have isolated peptides with antioxidant, antibacterial, and hypotensive activities from scallops. However, research on obtaining dual-target peptides with both ACE inhibitory and α-amylase inhibitory activities from scallop skirts based on systematic virtual screening combined with experimental verification is still rare. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a product derived from scallops ( Argopecten irradians The study focuses on a dual-target active peptide with both angiotensin-converting enzyme (ACE) and α-amylase inhibitory activities, along with its molecular simulation-based virtual screening, preparation method, and application in antihypertensive and hypoglycemic products.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a dual-target active peptide derived from the skirt of scallops, wherein the active peptide is selected from one of the following amino acid sequences: VDMFWR, PAGGR and SSCLR; preferably, the amino acid sequence of the active peptide is VDMFWR.

[0007] The present invention also provides a method for screening the above-mentioned active peptides, comprising the following steps: Step 1: Take fresh scallop skirts, add deionized water at a ratio of 1g:(2-4)mL, homogenize, add papain for enzymatic hydrolysis, inactivate the enzyme, centrifuge and take the supernatant to obtain the hydrolysate. Step 2: The enzymatic hydrolysate obtained in Step 1 is fractionated by ultrafiltration through an ultrafiltration centrifuge tube to obtain components with a molecular weight <3 kDa, which are then lyophilized for later use. Step 3: Perform LC-MS / MS peptidomics identification on the <3 kDa fraction obtained in Step 2 to obtain a set of polypeptide sequences; Step 4: The peptide sequences identified in Step 3 are used to predict bioactivity using PeptideRanker to screen peptides with a score > 0.6. Subsequently, online tools are used to predict ACE inhibitory activity, sensitization, water solubility, and toxicity to screen out candidate peptides that are non-toxic, have good water solubility, and have not been reported (the peptides obtained can be analyzed through the BIOPEP website to determine whether the peptides have been reported). Step 5: Perform molecular docking between the candidate peptides screened in Step 4 and the crystal structures of angiotensin-converting enzyme (ACE, PDB: 1O86) and α-amylase (PDB: 4GQR), respectively. Based on the binding energy, the number of hydrogen bonds and the binding mode with the active pocket, the dual-target active peptides with stable binding modes to both enzymes are finally screened out. The amino acid sequence of these peptides is at least one of VDMFWR, PAGGR and SSCLR.

[0008] Furthermore, the enzymatic hydrolysis conditions described in step 2 are as follows: papain addition of 4000-5000 U / g raw material, hydrolysis temperature of 50-60℃, and hydrolysis time of 3-5 hours.

[0009] The present invention also provides the use of the above-mentioned dual-target active peptide in the preparation of angiotensin-converting enzyme inhibitors and / or α-amylase inhibitors.

[0010] The present invention also provides the use of the above-mentioned dual-target active peptide in the preparation of a medicament for improving or treating hypertension and / or hyperglycemia-related conditions, wherein the active peptide is selected from at least one of VDMFWR, PAGGR or SSCLR.

[0011] Furthermore, the application is the use of active peptides in the preparation of reagents or products having at least one of the following functions: (1) Inhibits angiotensin-converting enzyme (ACE) activity; (2) Inhibits α-amylase activity; (3) Protects Ang II-induced endothelial cells and improves cell viability; (4) Reduce the content of endothelin-1 (ET-1) in Ang II-induced endothelial cells; (5) Promotes the release of nitric oxide (NO) from Ang II-induced endothelial cells; (6) Reduces the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in insulin-resistant HepG2 cells; (7) Increase the activity of hexokinase (HK) and pyruvate kinase (PK) in insulin-resistant HepG2 cells; (8) Reduce the levels of total cholesterol (TC) and triglycerides (TG) in insulin-resistant HepG2 cells.

[0012] The present invention also provides the use of the above-mentioned dual-target active peptide in the preparation of antihypertensive and / or hypoglycemic drugs.

[0013] Furthermore, the application includes using the active peptide as an active ingredient to prepare tablets, capsules, oral liquids, or powders.

[0014] The present invention also provides a composition for improving hypertension and / or insulin resistance-related conditions, comprising at least one of the above-mentioned active peptides, and an acceptable carrier or excipient.

[0015] Compared with the prior art, the advantages of the present invention are as follows: 1. First Discovery and Innovative Source: This invention is the first to identify and screen dual-target bioactive peptides with amino acid sequences VDMFWR, PAGGR, and SSCLR from the skirt of scallops. A search of databases such as BIOPEP revealed no previously reported peptides, classifying them as novel bioactive peptides. This invention achieves high-value utilization of scallop processing byproducts.

[0016] 2. Dual-target synergistic activity: The active peptide of this invention exhibits significant inhibitory activity against both ACE and α-amylase. Specifically, VDMFWR shows an IC50 value of [missing information - likely referring to an IC50 value] against both ACE and α-amylase. 50 The values ​​reached 0.327 mg / mL and 0.223 mg / mL, respectively, demonstrating excellent dual-target inhibition potential, which is superior to single-target inhibitors and more conducive to the comprehensive management of metabolic syndrome.

[0017] 3. Multiple Cellular Protection Mechanisms: At the cellular level, the active peptides provided by this invention not only exert a hypotensive effect by inhibiting ACE, but also comprehensively improve vascular function through multiple pathways, including antioxidation (scavenging ROS and MDA) and protection of vascular endothelium (regulating NO / ET-1 balance). Regarding blood glucose control, it can significantly improve glucose metabolism (increasing HK / PK activity) and lipid metabolism (reducing TC / TG) disorders under insulin resistance, exerting a multi-dimensional metabolic regulatory effect.

[0018] 4. High safety: Cytotoxicity experiments have confirmed that the three active peptides have no significant toxicity to EA.hy926 endothelial cells and HepG2 cells in the concentration range of 0-800 μg / mL, and have good biocompatibility, showing good prospects for development and application. Attached Figure Description

[0019] Figure 1 The degree of hydrolysis and A of scallop myosin heavy chain P24733·MYS_ARGIR were determined by virtual enzymatic hydrolysis of different proteases. E Value results graph; Figure 2 Endogenous fluorescence spectra of scallop skirt hydrolysates of different molecular weights; Figure 3 The activity analysis of scallop skirt enzymatic hydrolysates of different molecular weights is shown in Figure 1. 'a' represents ACE inhibitory activity, and 'b' represents α-amylase inhibitory activity. The same letter indicates no significant difference between the two groups, while different letters indicate significant differences between the two groups. Figure 4 The results show the molecular docking of three active peptides, where a is VDMFWR and 1O86, b is VDMFWR and 4GQR, c is PAGGR and 1O86, d is PAGGR and 4GQR, e is SSCLR and 1O86, and f is SSCLR and 4GQR. The yellow dashed lines represent hydrogen bonds, and the red, blue, and green lines in the middle represent peptide ligands. Figure 5 The safe concentration range for the cellular-level antihypertensive activity of functional peptides is given, where *** indicates P < 0.001 and ns indicates no significant difference. Figure 6 In the figure, 'a' represents the Ang II-induced hypertension injury model. The Gaussian amplitude model was used to perform nonlinear fitting of the data points to determine the peak position (xc≈1.15) and peak height. The coefficient of determination R0 is... 2 The value reached 0.9975, indicating that the model can accurately describe the variation of experimental data. b represents the protection of functional peptides against Ang II-damaged cells, and c represents the ACE inhibition rate of functional peptides. In the figure, ** indicates P < 0.01, *** indicates P < 0.001, ns indicates no significant difference, and GaussAmp represents the fitting function. Figure 7 The assay was performed to detect vasoactive substances and oxidative stress indicators after treatment with functional peptides, where a represents ET-1 content, b represents NO content, c represents ROS content, and d represents MDA content. ** indicates P < 0.01, and *** indicates P < 0.001. Figure 8 The safe concentration range of functional peptides with hypoglycemic activity at the cellular level, where *** indicates P < 0.001 and ns indicates no significant difference; Figure 9 To determine the optimal modeling concentration of HepG2 cells by inducing different concentrations of insulin using glucose consumption as the evaluation index, the same letter indicates no significant difference between the two groups, and different letters indicate significant differences between the two groups. Figure 10 The effect of functional peptides on key enzymes in glucose metabolism is shown, where a represents PK content and b represents HK content. Figure 11 The effects of functional peptides on lipid metabolism and oxidative stress are shown in the figure. a represents TC content, b represents TG content, c represents ROS content, and d represents MDA content. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference. In the above figure, the normal group refers to normal cells, the model group refers to model group cells, the control group refers to positive drug control group cells, the antihypertensive model group is cells induced by Ang II, and the hypoglycemic model group is cells induced by insulin. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0021] Example 1: Virtual enzymatic hydrolysis and protease screening of bioactive peptides from scallop skirt This embodiment aims to rapidly screen commercial proteases most suitable for releasing bioactive peptides from scallop myosin using computer-generated virtual enzymatic hydrolysis technology.

[0022] 1. Experimental Methods Obtain bay scallops from the UniProt database ( Argopecten irradians The amino acid sequence of the myosin heavy chain (accession number: P24733·MYS_ARGIR) was obtained. The protein sequence was virtually cleaved by pepsin (pH 1.3), trypsin, papain, and chymotrypsin using the "Potential Bioactivity" and "Virtual Enzymatic Digestion" tools of the BIOPEP-UWM database.

[0023] Based on the theoretical degree of hydrolysis (TDH) and the frequency of release of active peptides by specific enzymes (A... E The TDH value is used as an evaluation index. TDH reflects the protein cleavage efficiency of the protease; A EThe higher the value, the stronger the potential ability of the enzyme to release fragments with the target biological activities (ACE inhibition and α-amylase inhibition).

[0024] 2. Experimental Results The results are as follows Figure 1 As shown, among the four proteases tested, papain exhibited the best virtual enzymatic hydrolysis effect, with a theoretical degree of hydrolysis reaching 35.85%, and also showed the best performance in hydrolysis of antihypertensive and hypoglycemic peptides. E The values ​​were 0.0665 and 0.0452, respectively, both significantly higher than those of other proteases. Therefore, papain was selected as the optimal enzyme for subsequent enzymatic hydrolysis.

[0025] Example 2: Preparation and Ultrafiltration Fractionation of Active Peptides from Scallop Skirt 1. Preparation of enzymatic hydrolysis products Fresh scallop skirts were collected, cleaned, and impurities were removed. Deionized water was added at a ratio of 1g:3mL, and homogenized at 7500rpm for 0.3min using a homogenizer. The homogenate was then placed in an ice bath for later use. Papain (4500 U / g raw material) was added, and the mixture was enzymatically hydrolyzed in a 55℃ water bath for 4 hours. After hydrolysis, the enzyme was inactivated at 95℃ for 10min, cooled, and centrifuged (4℃, 10000rpm, 15min). The supernatant was collected as the scallop skirt hydrolysate. The degree of hydrolysis under these conditions was determined to be 36.02±1.51%, and the polypeptide content was 2.26±0.20 mg / mL, which highly matched the predicted values ​​from the virtual enzymatic hydrolysis analysis.

[0026] 2. Ultrafiltration grading The above enzymatic hydrolysate was sequentially passed through 3 kDa, 5 kDa, and 10 kDa ultrafiltration centrifuge tubes and centrifuged at 4500 rpm for 15 min. Three fractions with molecular weights of <3 kDa, 3-5 kDa, and 5-10 kDa were collected, respectively. Each fraction was lyophilized to obtain lyophilized powders of enzymatic hydrolysate products with different molecular weight ranges.

[0027] 3. Endogenous fluorescence spectroscopy analysis To verify the effects of enzymatic hydrolysis and ultrafiltration, an excitation wavelength of 292 nm was used, with both excitation and emission slit widths set to 5 nm, a scan rate of 1200 nm / min, and a voltage of 500 V. Endogenous fluorescence was measured for enzymatic hydrolysates of different molecular weights, and emission spectra in the 300-450 nm wavelength range were collected. The results are as follows: Figure 2 As shown, the maximum emission wavelength (λmax) of the 3-5 kDa and 5-10 kDa fractions shows a red shift compared to the <3 kDa fraction, indicating that ultrafiltration effectively separates peptides with different conformations and hydrophobicities. Aromatic amino acid residues in small molecular weight peptides are exposed to a more polar environment, resulting in a looser structure. This result confirms that large protein molecules are effectively degraded into small peptides during enzymatic hydrolysis.

[0028] Example 3: In vitro activity verification and active component locking of components with different molecular weights To rapidly identify the highly active components, the three molecular weight components obtained in Example 2 were prepared into 1 mg / mL solutions, and their ACE inhibitory activity was determined using the FAPGG method. The results are shown in Table 1 and... Figure 3 As shown in Figure a, the <3 kDa fraction had the highest ACE inhibition rate, reaching 51.88±6.44%, which was higher than that of the 3-5 kDa (44.45±4.81%) and 5-10 kDa (30.55±4.81%) fractions, but not significantly different from the 0.5 mg / mL captopril positive control (48.33±2.89%).

[0029] Using 5 mg / mL acarbose (51.70 ± 1.52%) as a control, the α-amylase inhibitory activity of 5 mg / mL lyophilized powder solutions of the three molecular weight fractions was determined. The results are shown in Table 1 and... Figure 3 As shown in Figure b, the <3 kDa fraction had the highest ACE inhibition rate, reaching 50.77±1.54%, which was significantly higher than that of the 3-5 kDa (31.79±4.23%) and 5-10 kDa (27.63±2.34%) fractions.

[0030] These results indicate that low molecular weight peptides are the main contributors to ACE inhibitory activity. Therefore, enzymatic digests with molecular weights <3kDa were targeted for subsequent peptide identification and virtual screening.

[0031] Table 1. ACE inhibition rate of different molecular weight components

[0032] Example 4: Peptidomics Identification and Bioinformatics Virtual Screening The <3 kDa fraction with the highest activity in Example 3 was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). After desalting and enrichment, the sample was analyzed using a nano-liquid chromatography-tandem Q-Exactive mass spectrometer. The obtained spectra were compared with the UniProt database, and a total of 1757 peptides were identified, of which 81.84% were short peptides composed of 3-6 amino acid residues, consistent with the high activity characteristics of low molecular weight fractions.

[0033] Multi-level virtual filtering To efficiently and accurately identify target bioactive peptides, a systematic virtual screening process was established: Initial activity screening: The identified peptide sequences were submitted to the PeptideRanker online tool, and 97 potentially highly active peptides were screened out with a score > 0.6 as the threshold.

[0034] ACE inhibitory activity prediction: The above peptides were input into the pLM4ACE prediction model, and 20 peptides with high predicted ACE inhibitory activity were screened out.

[0035] Safety and physicochemical property screening: AllerCatPro (allergenicity), Protein-Sol (water solubility), and ToxinPred (toxicity) were used sequentially for screening. Potential allergens, poorly water-soluble peptides, or toxic peptides were eliminated.

[0036] Novelty Confirmation: The five retained peptide sequences were searched in the BIOPEP database, and three of them were confirmed to be novel peptides that had not been reported before. Their sequences were VDMFWR, PAGGR, and SSCLR.

[0037] Example 5: Molecular docking verification and analysis of the dual-target inhibition mechanism To verify the reliability of the virtual screening results and to preliminarily elucidate the inhibition mechanism, the three novel peptides obtained in Example 4 were molecularly docked with the crystal structures ACE (PDB ID: 1O86) and α-amylase (PDB ID: 4GQR).

[0038] The three-dimensional structures of peptides were drawn and optimized using ChemDraw and Chem3D software. The crystal structures of the target protease were downloaded from the RCSB PDB database and pretreated using AutoDockTools, including dehydration and hydrogenation. Semi-flexible molecular docking was performed using AutoDock Vina, and binding energies were calculated and interaction modes analyzed.

[0039] The results are as follows Figure 4 As shown, the VDMFWR peptide exhibits low binding energies to both ACE (1O86) and α-amylase (4GQR), forming 9 and 5 hydrogen bonds, respectively. Importantly, VDMFWR connects to key residues in the S1 pocket of ACE's active site, indicating its ability to occupy the ACE active site and hinder substrate binding; its interaction site with α-amylase is far from the active pocket, suggesting a non-competitive inhibition mechanism. PAGGR's binding pattern to ACE covers key residues in the S1, S1', and S2 active pockets, demonstrating competitive inhibition; its binding site with α-amylase is far from the active site. SSCLR also shows stable binding to both target enzymes. The structural characteristics of the three peptides (such as the N-terminal valine of VDMFWR, the flexible fragment of PAGGR, and the amphiphilicity of SSCLR) are consistent with those of known active peptides. Based on the molecular docking results, all three peptides possess the potential to act as dual-target (ACE and α-amylase) inhibitors.

[0040] Example 6: Activity Verification and Inhibition Type Investigation of Synthetic Peptides To further verify the actual biological activity of the above three peptides, peptide fragments with a purity ≥95% (purchased from Hefei Guotai Biotechnology Co., Ltd.) were obtained by solid-phase synthesis, and their in vitro ACE inhibitory activity and α-amylase inhibitory activity were measured.

[0041] Half-maximal inhibitory concentration (IC50) 50 ) Measurement ACE inhibitory activity was determined using the FAPGG method, and α-amylase inhibitory activity was determined using the starch-iodine method. The results showed (see Table 2) that the three peptides VDMFWR, PAGGR, and SSCLR all exhibited significant ACE and α-amylase inhibitory activities.

[0042] Table 2. ACE and α-amylase IC50 of the three synthetic peptides 50 value

[0043] Example 7: Cellular-level hypotensive activity analysis of functional peptides from scallop skirt This embodiment aims to evaluate the hypotensive activity and mechanism of the three active peptides at the cellular level, using the human umbilical vein endothelial cell fusion cell line EA.hy926 as a model.

[0044] 1. Cytotoxicity assessment The effect of different concentrations (0-1600 μg / mL) of bioactive peptides on the viability of EA.hy926 cells was detected using the MTT assay. Results are as follows: Figure 5 As shown, within the concentration range of 0-800 μg / mL, the three peptides had no significant effect on cell viability, and this range was determined to be a safe concentration window. Subsequent studies were conducted within this concentration range.

[0045] 2. Establishment and protective effect of Ang II-induced hypertension injury model like Figure 6 As shown in Figure a, different concentrations of angiotensin II (Ang II) were used to induce damage in EA.hy926 cells, and 0.5 μM was determined to be the optimal concentration for modeling (IC50). 50 Within safe concentration ranges, cells were co-treated with AngII using three peptides (low, medium, and high concentrations). Results were as follows... Figure 6 As shown in Figures b and c, compared with the model group, treatment with all three peptides significantly improved the viability of damaged cells in a dose-dependent manner. With captopril as the positive control and the untreated group as the blank control, the results showed that captopril restored the ACE inhibition rate.

[0046] 3. Detection of vasoactive substances and oxidative stress indicators To further investigate the mechanism of action, key indicators in cell culture supernatant and cell lysate were detected, and the results are as follows: Figure 7As shown.

[0047] ET-1 (Endothelin-1): Endothelin is an endogenous vasomotor factor composed of 21 amino acids, possessing a sustained and potent vasoconstrictive function. Currently, three types of human endothelin have been identified: ET-1, ET-2, and ET-3, with ET-1 being the primary vasoconstrictor. Figure 7 As shown in Figure a, ET-1 levels significantly increased after Ang II-induced EA.hy926 cell damage, indicating enhanced vasoconstriction. The three peptides, at low, medium, and high concentrations, significantly reduced ET-1 levels in a concentration-dependent manner after treatment of damaged cells.

[0048] NO (nitric oxide): NO is a key signaling molecule in endothelial cells that regulates blood pressure. It is chemically reactive and highly lipid-soluble. It lowers blood pressure by reducing calcium ion concentration in endothelial cells, thereby causing vasodilation and increasing blood flow. Figure 7 As shown in Figure b, NO levels decreased significantly after Ang II stimulation, indicating impaired vasodilatory function; treatment with all three peptides significantly promoted NO production in a concentration-dependent manner.

[0049] ROS (Reactive Oxygen Species): Excessive ROS concentration can induce apoptosis and trigger cardiovascular diseases. For example... Figure 7 As shown in Figure c, Ang II can induce ROS production through pathways such as reduced coenzyme I / II, thus significantly increasing ROS levels in the model group. All three peptides exhibited ROS scavenging function at different concentrations.

[0050] MDA (malondialdehyde): MDA is an end product of lipid peroxidation. Higher levels of MDA indicate more severe oxidative damage to cell membranes and stronger oxidative stress. Figure 7 As shown in Figure d, Ang II induction significantly increased MDA content, and treatment with all three peptides effectively reduced MDA content (P<0.05), showing a concentration-dependent effect, with more significant effects at concentrations above 400 μg / mL.

[0051] The above results indicate that the active peptides from scallop skirts can not only directly inhibit ACE activity, but also exert a comprehensive hypotensive protective effect through multiple pathways such as antioxidation, protection of endothelial function, and regulation of NO / ET-1 balance.

[0052] The above results indicate that the active peptides from scallop skirts not only directly inhibit ACE activity but also exert a comprehensive antihypertensive protective effect through multiple pathways: on the one hand, they exert antioxidant effects by scavenging ROS and reducing MDA levels, thus alleviating oxidative stress and lipid peroxidation damage; on the other hand, they regulate the NO / ET-1 balance by reducing ET-1 and promoting NO production, protecting vascular endothelial function and maintaining vascular homeostasis. In summary, the active peptides from scallop skirts constitute a complete antihypertensive protective pathway, with antioxidation as the upstream link, endothelial function protection as the midstream hub, and NO / ET-1 balance regulation as the downstream effect.

[0053] Example 8: Cellular-level hypoglycemic activity analysis of functional peptides from scallop skirt This study used human hepatocellular carcinoma HepG2 cells and metformin as a positive control. An insulin resistance (IR-HepG2) model was established by inducing insulin resistance with high concentrations. The in vitro hypoglycemic activity of three bioactive peptides derived from scallop skirts was evaluated from the perspectives of cytotoxicity, glucose metabolism, lipid metabolism, and oxidative stress.

[0054] 1. Cytotoxicity and Model Establishment CCK-8 method verification as follows Figure 8 As shown, the three bioactive peptides exhibited no toxicity to HepG2 cells within a concentration range of 0-800 μg / mL, and the safe concentration range was clearly defined. Figure 9 As shown, using glucose consumption as the evaluation index, HepG2 cells were induced with different concentrations of insulin to determine the optimal levels of glucose consumption. -8 The optimal concentration for modeling was mol / L, and the IR-HepG2 cell model was successfully established.

[0055] 2. Effects on the activity of key enzymes in glucose metabolism The results are as follows Figure 10 As shown in Figures a and b, compared with the normal group, the activities of hexokinase (HK) and pyruvate kinase (PK) in the model group cells were significantly reduced (P<0.01), indicating that the glycolytic pathway was impaired and the cells' ability to take up and utilize glucose was reduced. Compared with the model group, treatment with the three active peptides significantly increased the activities of HK and PK (P<0.01). HK, as a key rate-limiting enzyme in glycolysis, catalyzes glucose phosphorylation to initiate metabolism; PK, as the rate-limiting enzyme at the end of glycolysis, catalyzes the conversion of phosphoenolpyruvate to pyruvate, promoting glucose breakdown for energy production. The results indicate that the three glucose-lowering peptides can effectively alleviate the glucose metabolism disorder in IR-HepG2 cells, enhance glucose consumption, and synergistically exert a glucose-lowering effect.

[0056] 3. Effects on lipid metabolism and oxidative stress The results are as follows Figure 11As shown in the ad, the levels of total cholesterol (TC), triglycerides (TG), reactive oxygen species (ROS), and malondialdehyde (MDA) in the model group were significantly higher than those in the normal group (P<0.01), confirming successful modeling and the presence of significant lipid deposition and oxidative stress damage. Compared with the model group, treatment with the three bioactive peptides reduced the above indicators to varying degrees. Among them, the bioactive peptide SSCLR showed the most significant effect in reducing TC and TG, and the positive control group also showed a highly significant improvement (P<0.01). The study shows that excessive lipid accumulation is a prerequisite for insulin resistance and β-cell dysfunction, and elevated TC and TG are strongly positively correlated with the prevalence and mortality of T2DM. Bioactive peptides regulate lipid metabolism and reduce lipid deposition by inhibiting the accumulation of TG and TC in IR-HepG2 cells, thereby improving insulin sensitivity and synergistically improving glucose metabolism.

[0057] In summary, using metformin as a positive control, the functional peptides from scallop skirts exhibited clear hypoglycemic activity at the cellular level. The mechanism encompasses two aspects: at the glucose metabolism level, by increasing the activity of HK and PK, it promotes cellular glucose uptake and glycolysis, thereby increasing glucose consumption; at the lipid metabolism and oxidative stress level, by reducing TC, TG, ROS, and MDA levels, it alleviates lipid deposition and oxidative damage, improving insulin resistance. These multi-pathway synergistic effects contribute to its in vitro hypoglycemic efficacy, providing cellular evidence for its potential application in type 2 diabetes intervention.

[0058] The cell experiment data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for statistical testing, and P < 0.05 was considered statistically significant.

[0059] Example 9: Compositions containing the active peptides of the present invention This embodiment provides a solid beverage composition for assisting in lowering blood pressure and blood sugar. By weight, it comprises: 5-10 parts of the active peptide VDMFWR described in this invention, and acceptable food additives such as 20-30 parts of maltodextrin, 10-20 parts of microcrystalline cellulose, 1-2 parts of citric acid, and 0.1-0.5 parts of steviol glycosides. The above components are mixed evenly, granulated, dried, and sized to obtain a granular solid beverage.

[0060] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A dual-target active peptide derived from the skirt of scallops, characterized in that: The amino acid sequence of the active peptide is VDMFWR.

2. A method for screening dual-target bioactive peptides derived from scallop skirts, characterized in that... Includes the following steps: Step 1: Take fresh scallop skirts, add deionized water at a mass-to-volume ratio of 1g:(2-4)mL, homogenize, add papain for enzymatic hydrolysis, inactivate the enzyme, centrifuge and take the supernatant to obtain the hydrolysate. Step 2: The enzymatic hydrolysate obtained in Step 1 is fractionated by ultrafiltration through an ultrafiltration centrifuge tube to obtain components with a molecular weight <3 kDa, which are then lyophilized for later use. Step 3: Perform LC-MS / MS peptidomics identification on the <3 kDa fraction obtained in Step 2 to obtain a set of polypeptide sequences; Step 4: The peptide sequences identified in Step 3 were used to predict bioactivity using PeptideRanker to screen peptides with a score > 0.

6. Subsequently, online tools were used to predict ACE inhibitory activity, sensitization, water solubility, and toxicity to screen out candidate peptides that are non-toxic, have good water solubility, and have not been reported before. Step 5: Perform molecular docking between the candidate peptides screened in Step 4 and the crystal structures of angiotensin-converting enzyme and α-amylase, respectively. Based on the binding energy, the number of hydrogen bonds and the binding mode with the active pocket, finally screen out dual-target active peptides that have a stable binding mode with both enzymes. Their amino acid sequences are at least one of VDMFWR, PAGGR and SSCLR.

3. The screening method according to claim 2, characterized in that: The enzymatic hydrolysis conditions described in step 2 are: papain addition of 4000-5000 U / g raw material, hydrolysis temperature of 50-60℃, and hydrolysis time of 3-5 hours.

4. The use of the dual-target active peptide of claim 1 or the dual-target active peptide prepared by the method of claim 2 in the preparation of angiotensin-converting enzyme inhibitors and / or α-amylase inhibitors.

5. The use of the dual-target active peptide of claim 1 or the dual-target active peptide prepared by the method of claim 2 in the preparation of a medicament for improving or treating hypertension and / or hyperglycemia-related conditions.

6. The application according to claim 5, characterized in that... The application refers to the use of active peptides in the preparation of reagents or products having at least one of the following functions: (1) Inhibits angiotensin-converting enzyme activity; (2) Inhibits α-amylase activity; (3) Protects Ang II-induced endothelial cells and improves cell viability; (4) Reduce the endothelin-1 content in Ang II-induced endothelial cells; (5) Promotes Ang II-induced nitric oxide release in endothelial cells; (6) Reduces reactive oxygen species and malondialdehyde levels in insulin-resistant HepG2 cells; (7) Increase the activity of hexokinase and / or pyruvate kinase in insulin-resistant HepG2 cells; (8) Reduce the levels of total cholesterol and / or triglycerides in insulin-resistant HepG2 cells.

7. The use of the dual-target active peptide of claim 1 or the dual-target active peptide prepared by the method of claim 2 in the preparation of antihypertensive and / or hypoglycemic drugs.

8. The application according to claim 7, characterized in that: The applications include using the active peptides as active ingredients to prepare tablets, capsules, oral liquids, or powders.

9. A composition for improving hypertension and / or insulin resistance-related conditions, characterized in that, It comprises at least one of the dual-target active peptides prepared by the method of claim 2, and an acceptable carrier or excipient.

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