A castor moth pupa hydrolysate and a screening method of active peptides thereof

Abstract

This invention discloses a method for screening the hydrolysate of castor silkworm pupae and its bioactive peptides. The hydrolysate contains several α-glucosidase inhibitory peptides, with Tyr-Tyr-Leu-Glu-Arg showing the best effect. The screening method involves: extracting castor silkworm pupa protein using an alkaline extraction and acid precipitation method; screening enzymes for enzymatic hydrolysis based on α-glucosidase inhibition effect; separating the enzymatic hydrolysate by ultrafiltration; and using peptidomics, gastrointestinal enzyme simulated digestion, and multidimensional virtual screening to screen for target α-glucosidase inhibitory peptides with good hypoglycemic effects and good gastrointestinal digestion and absorption. The mechanism of action is explored using isothermal titration microcalorimetry and molecular dynamics. This invention simplifies the traditional peptide preparation process and effectively improves the preparation efficiency of target peptides. The screened target peptides show significantly better α-glucosidase inhibition effects than previously reported food-derived peptides, and have broad market prospects in the resource utilization of castor silkworm pupae and the development of novel hypoglycemic peptides.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a method for screening castor silkworm pupa hydrolysate and its active peptides. Background Technology

[0002] Type 2 diabetes is a metabolic disease caused by insulin resistance and pancreatic β-cell dysfunction. Its core characteristic is persistent hyperglycemia, accompanied by metabolic disorders of the three major nutrients: carbohydrates, proteins, and fats. Long-term uncontrolled hyperglycemia can continuously damage multiple tissues and organs throughout the body, inducing various chronic complications such as diabetic nephropathy and cardiovascular disease, seriously threatening patients' lives and health, and has become a major global public health problem. Inhibiting α-glucosidase (α-GC) activity is one of the key approaches in the clinical treatment of type 2 diabetes. This mechanism can stabilize postprandial blood glucose levels by delaying the breakdown and absorption of carbohydrates in the intestine. Currently, commonly used chemically synthesized α-glucosidase inhibitors such as acarbose, voglibose, and miglitol, while effective in lowering blood sugar, pose several safety risks with long-term use: First, the risk of liver damage is significant. Acarbose easily induces asymptomatic liver enzyme elevations, with an incidence rate of 2%-5%, and voglibose has occasionally been associated with severe liver dysfunction accompanied by elevated AST / GPT. Second, cardiovascular risks are exacerbated. Improper use of acarbose can lead to significant fluctuations in blood sugar, damage to vascular endothelium, and induce coronary artery spasm, increasing the risk of serious cardiovascular events such as myocardial infarction by 28%. Third, systemic adverse reactions are frequent. Voglibose may cause facial edema, and long-term use of miglitol can hinder intestinal iron absorption, leading to iron deficiency anemia. Therefore, the development of safe, non-toxic, and low-side-effect natural α-glucosidase inhibitors has become a research hotspot in the field of diabetes prevention and treatment.

[0003] In the research and development of natural active ingredients, food-derived bioactive peptides have become ideal research subjects for α-glucosidase inhibitors due to their advantages such as high safety, easy absorption, and diverse bioactivities. Among them, edible insects, as high-quality food protein resources, have extremely high development value: their protein content generally reaches more than 40%, and some species even exceed 80%, with a complete and balanced composition of essential amino acids that can meet the nutritional needs of the human body; at the same time, insect protein can produce bioactive peptides with various physiological activities such as anti-diabetic and antioxidant effects after enzymatic hydrolysis. In addition, insect farming has the characteristics of low resource consumption and environmental sustainability, making it a high-quality source for exploring food-derived α-glucosidase inhibitory peptides (α-GIPs). Lepidoptera, the second largest order in the class Insecta, accounts for one-fifth of all edible insect species, offering significant advantages for industrial development. They possess high biomass, short reproductive cycles, and low feeding costs. Their protein content reaches 40%-70% of dry weight, with a balanced amino acid composition. Furthermore, their enzymatic hydrolysates are mostly small-molecule bioactive peptides, exhibiting characteristics of small molecular weight, easy absorption, and low toxicity, making them promising for applications in functional foods and medicine. In particular, the silkworm (Bombyx mori), as a model insect, has been extensively reported to possess ACE inhibitory, antioxidant, and immunomodulatory activities in its hydrolysates and peptides. A few reports have also documented α-GC inhibitory activity; Luo et al. demonstrated α-GC inhibitory activity after hydrolyzing silkworm pupa protein with pepsin and bromelain, with an IC50 value of [missing information]. 50 The concentrations were 9.520 mg / mL and 19.597 mg / mL, respectively. Xun et al. further isolated a novel peptide after irradiation-assisted hydrolysis of silkworm pupa protein, and the IC50 of its α-GC inhibitory activity was [not specified]. 50 The concentration reached 446.17 ± 11.39 µg / mL.

[0004] As a representative non-model organism of the Saturniidae family in the Lepidoptera order, the castor silkworm (Samia ricini) has been farmed on a large scale in China, India, Japan, and other regions due to its strong adaptability and diverse feeding habits. The castor silkworm pupa (Samia ricini Pupa, SRP) has a stable source and a short rearing cycle, facilitating large-scale production. With a protein content as high as 50-55% and a balanced amino acid composition, it is theoretically a highly promising high-quality protein resource, providing an important direction for increasing its added value. However, in the existing silk processing chain, SRP is often discarded as a byproduct or treated only as low-value feed, resulting in significant resource waste. It is noteworthy that although Samia ricini is closely related to Bombyx mori, the two differ significantly in biological characteristics such as body size, diet, and metabolism, suggesting that SRP protein may contain unique structural features and enzymatic activity potential. However, due to the scarcity of non-model organism proteome databases, traditional database search strategies are insufficient to fully cover their unique peptide profile information. Currently, castor silkworm pupae are mostly discarded or used as low-value feed, and their development potential has not been fully realized. Their value as a source of α-glucosidase inhibitory peptides urgently needs to be explored in depth.

[0005] Currently, research on α-glucosidase inhibitory peptides derived from castor silkworm pupae remains insufficient, primarily due to a disconnect between experimental preparation and virtual screening. In terms of preparation processes, existing studies largely focus on crude extraction and enzymatic hydrolysis, lacking a standardized process from freeze-drying and defatting to purification and ultrafiltration fractionation (<3 kDa). This results in low enrichment of active ingredients and difficulty in obtaining samples suitable for high-resolution mass spectrometry analysis, impacting the reproducibility of the preparation. Regarding screening methods, while molecular docking and ADMET prediction are relatively mature, integrated studies combining "De novo peptidomics, multidimensional virtual screening, and gastrointestinal stability verification" are still lacking. This limits existing research to the crude extract level, lacking in-depth analysis of specific sequences, inhibitory mechanisms, and anti-digestion capabilities, hindering the industrial production of highly active peptides and the precise development of functional foods.

[0006] In summary, the breakthrough in the efficient and precise screening technology for α-glucosidase inhibitory peptides derived from castor silkworm pupae solves the technical bottlenecks in existing preparation and research. This not only fills the research gap in this field but also promotes the high-value utilization of castor silkworm pupae resources, providing a new pathway for the development of natural hypoglycemic drugs for type 2 diabetes. It has significant scientific research and industrial application value. Summary of the Invention

[0007] The purpose of this invention is to provide a method for screening castor silkworm pupa hydrolysates and their bioactive peptides. Given the high diversity of protein hydrolysates from different insect sources, a major challenge in the field of insect bioactive peptide research is how to establish efficient targeted screening strategies to identify α-glucosidase inhibitory peptides that possess both high inhibitory activity and good safety, and to develop scalable preparation and purification processes to achieve an optimal balance between purity and yield. This invention solves these problems by tailoring screening and separation strategies specifically for castor silkworm pupae.

[0008] To solve the above-mentioned technical problems, the first solution provided by the present invention is a castor silkworm pupa hydrolysate containing at least one α-glucosidase inhibitory peptide with an amino acid sequence of Tyr-Tyr-Leu-Glu-Arg, Thr-Asp-Pro-Ala-Phe, or Pro-Pro-Glu-Phe.

[0009] In some embodiments, the amino acid sequence of the α-glucosidase inhibitory peptide contained in the castor silkworm pupa hydrolysate is Tyr-Tyr-Leu-Glu-Arg.

[0010] To solve the above-mentioned technical problems, the second solution provided by the present invention is a method for screening active peptides in castor silkworm pupa hydrolysate. This screening method is used to screen for α-glucosidase inhibitory peptides contained in the castor silkworm pupa hydrolysate of the aforementioned first solution, and includes the following steps: S1, defatted castor silkworm pupa powder: freeze-dried castor silkworm pupa powder is mixed with n-hexane at a ratio of 1:(4-6) (g / mL) at 20-30℃, then centrifuged several times at 4℃ and the supernatant is discarded. After drying, defatted castor silkworm pupa powder is obtained.

[0011] S2, Preparation of castor silkworm pupa protein: Defatted castor silkworm pupa powder was mixed with distilled water, and the pH was adjusted to 10.5-11.5 with NaOH. Then, the pH was adjusted to 4.0-5.0 with HCl and stirred evenly. The mixture was centrifuged at 4°C, and the resulting precipitate was dialyzed and freeze-dried to obtain castor silkworm pupa protein.

[0012] S3, Castor silkworm pupa protein hydrolysis: Castor silkworm pupa protein and distilled water were mixed evenly at a ratio of 1:(8-12) (g / mL). The pH of the solution was adjusted to 7.0-8.0 with NaOH. 600 U / mL trypsin was added at 45-50℃ and the reaction was carried out with constant temperature shaking for 3-5 hours. The reaction was then terminated by heating in a 95℃ water bath for 15 minutes to inactivate the enzyme. The solution was centrifuged at 4℃, the supernatant was separated and freeze-dried to obtain castor silkworm pupa trypsin hydrolysate.

[0013] S4, Ultrafiltration Fractionation and Component Identification of Castor Silkworm Pupa Peptides: Castor silkworm pupa protein hydrolysate was reconstituted in distilled water at a ratio of 1:15 (g / mL), and fractionated using a centrifugal ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected and freeze-dried to obtain castor silkworm pupa-derived active peptide components with a molecular weight less than 3 kDa. The castor silkworm pupa-derived active peptide components were pretreated by dissolution, reductive alkylation, and SP2 desalting. The molecular weight distribution, amino acid composition, peptide chain length, and N / C-terminal amino acid types of the castor silkworm pupa-derived active peptide components were detected and identified using liquid chromatography-mass spectrometry.

[0014] S5, Screening of α-glucosidase inhibitory peptides: First, the active peptide components derived from castor silkworm pupae were subjected to pLM4Alg and ToxinPred 3.0 platform to remove sensitizing / toxic sequences, and the remaining candidate peptides were used in the molecular docking process; the ligand molecular structures were constructed using ChimeraX 1.10 and saved in PDBQT format, and molecular docking analysis was performed using AutoDock 4.2 to screen for high-affinity candidate peptides; the high-affinity candidate peptides were simulated in the gastrointestinal digestive environment using BIOPEP-UWM to analyze the undegraded peptides or newly generated active fragments after enzymatic hydrolysis, and the gastrointestinal absorption rate of the peptides was predicted using SwissADME; the candidate peptides were ranked based on three priority indicators: absolute binding energy, number of anti-digestive fragments, and absorption potential. The undegraded candidate peptides obtained were the active peptides of castor silkworm pupae hydrolysate, which are the α-glucosidase inhibitory peptides derived from castor silkworm pupae.

[0015] In some embodiments, step S1 is as follows: fresh castor silkworm pupae are washed, killed with liquid nitrogen, freeze-dried, and ground through an 80-mesh sieve; then the freeze-dried castor silkworm pupae powder is mixed with n-hexane at a ratio of 1:5 (g / mL), stirred at 400 rpm for 4 h at 25°C, centrifuged at 4000 rpm for 10 min at 4°C and the supernatant is discarded. The centrifugation operation is repeated three times, and defatted castor silkworm pupae powder is obtained after being dried with nitrogen.

[0016] In some embodiments, step S2 is as follows: defatted castor silkworm pupa powder and distilled water are mixed evenly at a ratio of 1:10 (g / mL), the pH is adjusted to 11.0 with 1 mol / L NaOH, and after 6 h, the pH is adjusted to 4.5 with 1 mol / L HCl and stirred for 4 h. The mixture is then centrifuged at 10,000 rpm for 15 min at 4 °C. The precipitate is dialyzed and freeze-dried to obtain castor silkworm pupa protein.

[0017] In some embodiments, step S3 is as follows: castor silkworm pupa protein and distilled water are mixed evenly at a ratio of 1:10 (g / mL), the pH of the solution is adjusted to 7.5 with 1 mol / L NaOH, 600 U / mL trypsin is added at 47°C and the reaction is carried out with constant temperature shaking for 4 h, then the reaction is terminated by heating in a water bath at 95°C for 15 min, the supernatant is separated by centrifugation at 10000 rpm for 15 min at 4°C, and the castor silkworm pupa trypsin hydrolysate is obtained after freeze drying.

[0018] In some implementations, step S4 is specifically as follows: S41, the castor silkworm pupa protein hydrolysate was reconstituted in distilled water at a ratio of 1:10-20 (g / mL). The supernatant after reconstitution was taken and added to a 3 kDa ultrafiltration tube. The mixture was centrifuged at 10,000 rpm for 15 min at 4℃. Peptide fractions with a molecular weight less than 3 kDa were collected from the permeate. The permeate was collected and freeze-dried to obtain active peptide fractions derived from castor silkworm pupae with a molecular weight less than 3 kDa.

[0019] S42, the active peptide components derived from castor silkworm pupae were pretreated by dissolution, reductive alkylation, and SP2 desalting, and then detected using an Easy-nLC 1200 / QExactive liquid chromatography-mass spectrometry system. Liquid chromatography conditions: pre-column 150 μm id × 50 mg / mL, analytical column 150 μm id × 170 mg / mL, mobile phase A 0.1% FA aqueous solution, mobile phase B 0.1% FA-80% acetonitrile solution, flow rate 600 nL / min, analysis time 66 min; mass spectrometry conditions: acquisition mode DDA, primary mass spectrometry resolution 70000, AGC target 3e6, scan range 100-1500 m / z, secondary mass spectrometry resolution 17500, AGC target 1e5, NCE 28.

[0020] S43. Liquid chromatography-tandem mass spectrometry (LCMS / MS) was used to detect trypsin hydrolysates from castor silkworm pupae with molecular weights <3 kDa. PEAKS de novo peptide sequencing technology was used to analyze the raw mass spectrometry data. Without relying on existing proteome databases, the amino acid sequence information of the active peptide components derived from castor silkworm pupae was resolved, identified, and obtained from the mass spectrometry fragment information, including molecular weight distribution, amino acid composition, peptide chain length, and N / C-terminal amino acid residue types. The search parameters were set as follows: fixed modification was urea methylation (Carbamidomethyl, C), variable modifications were oxidation (M) and acetylation (Acetyl, Peptide N-term), the enzyme digestion type was non-specific, the precursor ion mass tolerance was 20 ppm, and the fragment ion mass tolerance was 0.02 Da.

[0021] In some implementations, step S5 is specifically as follows: S51, after removing sensitizing / toxic sequences from the active peptide components derived from castor silkworm pupae using the pLM4Alg and ToxinPred 3.0 platforms, the remaining candidate peptides are used in the molecular docking process.

[0022] S52, the ligand molecular structure was constructed using ChimeraX 1.10 and saved in PDBQT format. Molecular docking analysis was performed using AutoDock 4.2 to screen for candidate peptides with high affinity. The specific parameters for molecular docking analysis were as follows: the grid center coordinates (center_x, center_y, center_z) were set to [3.250, -8.25, -2.556], the grid center was set at the enzyme active site (60×60×60 Å, spacing 0.375 Å), the genetic algorithm was run 10 times, the maximum energy evaluation was 2.5 million times, and the binding free energy ΔG < -7.5 kcal / mol was used as the threshold.

[0023] S53 uses BIOPEP-UWM to simulate the gastrointestinal digestive environment of high-affinity candidate peptides, analyzes undegraded peptides or newly generated active fragments after enzymatic hydrolysis, and combines SwissADME to predict the gastrointestinal absorption rate of peptides; the conditions of the simulated gastrointestinal digestive environment are: pepsin pH 3.0, trypsin / chymotrypsin pH 7.6, 37°C.

[0024] S54, based on three priority indicators—absolute binding energy, number of anti-digestion fragments, and absorption potential—ranked candidate peptides. The undegraded candidate peptides obtained were the active peptides of castor silkworm pupa hydrolysate, namely, α-glucosidase inhibitory peptides derived from castor silkworm pupae.

[0025] To solve the above-mentioned technical problems, the third solution provided by the present invention is an α-glucosidase inhibitor, which includes the active peptides of castor silkworm pupa hydrolysate obtained by the screening method in the first solution or the castor silkworm pupa hydrolysate obtained by the screening method in the second solution.

[0026] The application of castor silkworm pupa hydrolysate in the first solution or α-glucosidase inhibitor in the third solution in the preparation of antihypertensive drugs.

[0027] The advantages of this invention, which differ from existing technologies, are: This invention establishes a high-throughput screening system, simplifying the traditional peptide preparation process and improving the preparation efficiency and yield of target peptides, providing a technical path for the industrial development of novel hypoglycemic peptides. Specifically, this invention uses castor silkworm pupae as raw material. After defatting with n-hexane, protein is extracted from the castor silkworm pupae using an alkaline extraction and acid precipitation method. The protein is then enzymatically digested using a screened protease. The hydrolysate is separated by ultrafiltration and screened for α-glucosidase inhibitory peptides using ultra-high performance liquid chromatography-mass spectrometry, bioinformatics analysis, and molecular docking, filling the research gap in α-glucosidase inhibitory peptide sequences derived from castor silkworm pupae. Furthermore, the inhibitory effect (IC50) of specific small molecule peptides derived from castor silkworm pupae on α-glucosidase is also investigated. 50 The concentration (0.24 ± 0.05 mg / mL) was superior to the inhibitory activity (IC50) of taro globulin against α-glucosidase reported in previous literature. 50 The inhibitory activity (IC50) of specific secondary metabolites isolated from *Ballis* species (such as *Ballis prostrata* and *Ballis dentata*) against α-glucosidase was 2.09 ± 0.19 mg / mL. 50 (0.296 mg / mL / 0.568 mg / mL) (Ma Erlan, Lin Ying, Tu Lian, et al. Extraction and purification of taro globulin and its inhibitory activity against α-amylase and α-glucosidase [J]. Food Industry Technology, 2021, 42(14):25-32. DOI: 10.13386 / j.issn1002-0306.2020100266; Deng Zhentao. Study on chemical composition and bioactivity of four species of *Ballisneria* and *Diospyros kaki* [D]. Kunming: Kunming Institute of Botany, Chinese Academy of Sciences, 2025.), The α-glucosidase inhibitory peptide obtained from castor silkworm pupae has the characteristics of small molecular weight, easy absorption and low toxicity, which will significantly improve the utilization rate and added value of castor silkworm pupae, and has broad market prospects and industrial application value in the resource transformation, recycling and high-value utilization of castor silkworm pupae. Attached Figure Description

[0028] Figure 1 This is a peptide information diagram of the trypsin hydrolysate (MW < 3 kDa) in Example 1 of the present invention; wherein, (A) represents the molecular weight (MW) distribution, divided into the ranges of <1 kDa, 1 - 1.5 kDa and 1.5 - 3 kDa; (B) represents the frequency distribution of peptide chain length; (C) represents the total amino acid composition of the identified peptide; (D) represents the terminal amino acid residue analysis, showing the distribution of specific amino acid types (nonpolar aliphatic, aromatic, polar uncharged, basic and acidic) at the C-terminus (left) and N-terminus (right). Figure 2 This is a schematic diagram of the screening steps for α-glucosidase inhibitory peptides in Example 1 of the present invention; wherein, the green checkmark and the red cross represent the inclusion criteria and exclusion criteria, respectively; Figure 3 This is a two-dimensional schematic diagram of the interaction between the three peptides screened in Example 1 of the present invention and the non-specific sites of α-glucosidase; namely YYLER, TDPAF and PPEF; Figure 4 This is a dose-response curve of the trypsin hydrolysate (MW < 3 kDa) and the three peptides screened in Example 1 of this invention; wherein, (A) represents the <3 kDa hydrolysate and synthetic peptide; (B) represents YYLER, (C) represents TDPAF, and (D) represents the inhibitory dose-response curve of PPEF on α-glucosidase; the blue solid line is the nonlinear regression fitting curve (i.e., inhibitor concentration-response fitting), and the pink shaded area is the 95% confidence interval; the horizontal dashed line represents the 50% inhibition level (IC50). 50 (The blue dots represent experimental data points.) Figure 5 This is a kinetic analysis diagram of the enzyme inhibition of the three peptides screened in Example 1 of this invention; where YYLER (A-C), TDPAF (D-F), and PPEF (G-I) are Lineweaver-Burk plots (left column: A, D, G), Dixon plots (middle column: B, E, H), and Cornsh-Bowden plots (right column: C, F, I) for α-glucosidase inhibition; v is the initial reaction rate, [S] is the substrate (pNPG) concentration, and [I] is the inhibitor concentration; the inhibitor concentration is shown in the legend of each panel; the line represents the linear regression fit of the experimental data; Figure 6 This is a thermodynamic analysis diagram of the peptide YYLER screened in Example 1 of the present invention; wherein, (A) represents the original thermogram of heat flow (differential power) changing with time during titration; (B) represents the enthalpy change curve of each mole of injecting agent (ΔH) as a function of the molar ratio of YYLER to α-GC, plotted by integrating isotherms. Figure 7 This is a schematic diagram of the binding mechanism and structural characterization of the peptide YYLER screened in Example 1 of this invention; where (A) represents the energy decomposition analysis of the top 10 energy hotspots (residues), the net binding energy is decomposed into molecular mechanical interaction energy, polar solvation energy penalty and nonpolar solvation energy, and the error bars represent the standard deviation; (B) represents the representative three-dimensional (3D) binding mode extracted from the global lowest energy point of the free energy surface; (C) represents the free energy surface of the complex constructed using the first two principal components (PC1 and PC2) extracted from the 280-300 ns simulated trajectory, the blue area represents the lowest energy basin (metastable state), and the local magnified view highlights the key interactions, including the hydrogen bonds (black dashed lines) and salt bridges formed between the peptide (orange bar model) and the key enzyme residues (cyan bar model); Figure 8 This is a schematic flowchart illustrating the method and mechanism for screening active peptides from castor silkworm pupa hydrolysate according to the present invention. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the invention, are intended to cover non-exclusive inclusion.

[0031] In this invention, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0032] I. Screening Method Example 1

[0033] The screening steps for α-glucosidase inhibitory peptides derived from castor silkworm pupae in this embodiment are as follows: (1) Degreased castor silkworm pupa powder Fresh castor silkworm pupae were washed, killed with liquid nitrogen, freeze-dried, and ground through an 80-mesh sieve. The freeze-dried castor silkworm pupa powder was then mixed with n-hexane at a ratio of 1:5 (g / mL), stirred at 400 rpm for 4 h at 25°C, centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was discarded. The centrifugation operation was repeated three times, and the powder was dried with nitrogen to obtain defatted castor silkworm pupa powder.

[0034] (2) Preparation of castor silkworm pupa protein: Defatted castor silkworm pupa powder was mixed with distilled water at a ratio of 1:10 (g / mL). The pH was adjusted to 11.0 with 1 mol / L NaOH. After 6 h, the pH was adjusted to 4.5 with 1 mol / L HCl and stirred for 4 h. The mixture was then centrifuged at 10,000 rpm for 15 min at 4 °C. The precipitate was dialyzed and lyophilized to obtain castor silkworm pupa protein. The protein content (purity ≥95%) was determined using a Kjeltec™ 8400 analyzer (FOSS Analytics A / S; Hillerod, Denmark). The protein was stored at -80 °C for later use.

[0035] (3) Protein hydrolysis of castor silkworm pupae Castor silkworm pupa protein and distilled water were mixed evenly at a ratio of 1:10 (g / mL). The pH of the solution was adjusted to 7.5 with 1 mol / L NaOH. 600 U / mL trypsin was added at 47℃ and the reaction was carried out with constant temperature shaking for 4 h. The reaction was then terminated by heating in a water bath at 95℃ for 15 min. The supernatant was separated by centrifugation at 10000 rpm for 15 min at 4℃. Castor silkworm pupa trypsin hydrolysate was obtained after freeze drying.

[0036] (4) Ultrafiltration fractionation and component identification of castor silkworm pupa polypeptides The castor silkworm pupa protein hydrolysate was reconstituted in distilled water at a ratio of 1:15 (g / mL). The supernatant after reconstitution was taken and added to a 3 kDa ultrafiltration tube. The mixture was centrifuged at 10,000 rpm for 15 min at 4 °C. Peptide fractions with a molecular weight less than 3 kDa were collected from the permeate. The permeate was collected and freeze-dried to obtain active peptide fractions derived from castor silkworm pupae with a molecular weight less than 3 kDa.

[0037] The active peptides derived from castor silkworm pupae were pretreated by dissolution, reductive alkylation, and SP2 desalting, and then detected using an Easy-nLC 1200 / QExactive liquid chromatography-mass spectrometry system (Thermo Fisher Scientific). Liquid chromatography conditions: pre-column 150 μm id × 50 mg / mL (Reprosil-Pur 120 C18-AQ 3 μm), analytical column 150 μm id × 170 mg / mL (Reprosil-Pur 120 C18-AQ 1.9 μm), mobile phase A 0.1% FA aqueous solution, mobile phase B 0.1% FA-80% acetonitrile solution, flow rate 600 nL / min, analysis time 66 min. Mass spectrometry conditions: acquisition mode DDA, primary mass spectrometry resolution 70000, AGC target 3e6, scan range 100-1500 m / z, secondary mass spectrometry resolution 17500, AGC target... 1e5, NCE 28.

[0038] Raw mass spectrometry data were analyzed using PEAKS De novo sequencing technology. A strategy was employed to identify and obtain peptide sequence information of trypsin hydrolysate (MW < 3 kDa), including molecular weight distribution, amino acid composition, peptide chain length, and N / C-terminal amino acid types. This castor silkworm pupa-derived bioactive peptide component contained all 20 protein amino acids, with most peptides having a molecular weight < 1 kDa and lengths concentrated between 4 and 10 amino acid residues. The N-terminus was enriched with nonpolar aliphatic and aromatic amino acids, while the C-terminus was highly enriched with basic amino acids. The search parameters were: fixed modification as Carbamidomethyl (C), variable modifications as Oxidation (M) and Acetyl (Peptide N-term), enzyme type as Non-specific, precursor ion mass tolerance of 20 ppm, and fragment ion mass tolerance of 0.02 Da.

[0039] (5) Screening of α-glucosidase inhibitory peptides A systematic screening strategy was employed to evaluate α-glucosidase inhibitory peptides. First, after removing sensitizing / toxic sequences from 4,392 peptides using the pLM4Alg and ToxinPred 3.0 platforms, 1,100 candidate peptides were retained for molecular docking. Subsequently, the ligand molecular structure was constructed using ChimeraX 1.10 (UCSF RBVI, San Francisco, CA, USA) and saved in PDBQT format. Molecular docking analysis was performed using AutoDock 4.2 (Scripps Research Institute, La Jolla, CA, USA). The specific parameters were: grid center coordinates (center_x, center_y, center_z) were set to [3.250, -8.25, -2.556], the grid center was set at the enzyme active site (60×60×60 Å, spacing 0.375 Å), the genetic algorithm was run 10 times, and the maximum energy evaluation was 2.5 million times. Forty high-affinity candidate peptides were screened with a binding free energy ΔG < -7.5 kcal / mol as the threshold.

[0040] Furthermore, the gastrointestinal digestive environment (pepsy pH 3.0, trypsin / chymotrypsin pH 7.6, 37°C) was simulated using BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / en / biopep-uwm-2) to analyze undegraded peptides or newly generated active fragments after enzymatic hydrolysis, and the gastrointestinal absorption rate of peptides was predicted using SwissADME (http: / / www.swissadme.ch / ) (GI absorption="High" indicates high absorption potential).

[0041] The candidate peptides were finally ranked according to three priorities: (1) absolute binding energy (i.e., exhibiting inhibitory activity, in descending order); (2) number of anti-digestion fragments (i.e., exhibiting anti-digestion ability, in descending order); and (3) absorption potential (i.e., exhibiting absorption ability, in descending order). The novelty of the screened peptides was verified using the BIOPEP (www.uwm.edu.pl / biochemia), PeptideDB (www.peptides.be), and EROP-Moscow (erop.inbi.ras.ru) databases. Finally, three peptides were found to be undegraded after digestion, indicating that they had anti-digestion potential, i.e., α-glucosidase inhibitory peptides derived from castor silkworm pupae were screened. The three peptides were denoted as YYLER, TDPAF, and PPEF, respectively.

[0042] Comparative Example 1 Based on the preparation steps of Example 1, the only difference is that in step (3), trypsin is replaced with alkaline protease, and the other steps are the same as in Example 1.

[0043] Comparative Example 2 Based on the preparation steps of Example 1, the only difference is that in step (3), trypsin is replaced with papain, and the other steps are the same as in Example 1.

[0044] Comparative Example 3 Based on the preparation steps of Example 1, the only difference is that in step (3), trypsin is replaced with neutral protease, and the other steps are the same as in Example 1.

[0045] Comparative Example 4 Based on the preparation steps of Example 1, the only difference is that in step (3), trypsin is replaced with flavor protease, and the other steps are the same as in Example 1.

[0046] Comparative Example 5 Based on the preparation steps of Example 1, the only difference is that in step (3), trypsin is replaced with complex protease, and the other steps are the same as in Example 1.

[0047] Comparative Example 6 Based on the preparation steps of Example 1, the only difference is that in step (3), trypsin is replaced with pepsin, and the other steps are the same as in Example 1.

[0048] II. Testing and Analysis (1) Identification of α-glucosidase inhibition rate of trypsin hydrolysate from castor silkworm pupae The inhibition rate of α-glucosidase was determined using the substrate (pNPG) depletion method, and the half-inhibitory concentration (IC50) was calculated based on the dose-response curve. 50Enzymes were screened for enzymatic digestion. α-glucosidase solution (1 U / mL, dissolved in 0.1 M, pH 6.8, PBS) was mixed with the sample (1:1, v:v) and incubated at 37℃ for 20 min. Then, 50 μL of α-p-nitrophenylglucopyranoside (α-pNPG, 3.0 mg / mL) was added as a substrate. The reaction mixture was incubated at 37℃ for 5 min, and then 100 μL of 0.5 M Na2CO3 was added to terminate the reaction. The absorbance of released p-nitrophenol (NPG) was measured at 405 nm using a Molecular Devices SpectraMax i3x microplate reader (Molecular Devices, LLC; San Jose, CA, USA), and the α-glucosidase inhibition rate was calculated using the following expression. A dose-response curve was plotted with sample concentration on the x-axis and α-glucosidase inhibition rate on the y-axis, and the half-inhibitory concentration (IC50) was calculated. 50 ).

[0049] (1-1) In equation (1-1), A1 represents the absorbance value of the experimental sample group, A0 represents the absorbance value of the blank control group, and A S A represents the absorbance value of the enzyme background group. b This indicates the absorbance value of the sample background group.

[0050] (2) Identification of α-glucosidase inhibitory peptides derived from castor silkworm pupae To further elucidate the reasons for the high activity of castor silkworm pupa trypsin hydrolysate (SCRP protein trypsin hydrolysate), given the lack of public database annotations for SCRP protein, this invention employs PEAKS De novo sequencing technology to analyze raw mass spectrometry data and strategically identifies peptide sequences in the trypsin hydrolysate (MW < 3 kDa).

[0051] Please see Figures 1-3 ,in, Figure 1 This is a sequence diagram of peptide sequences from the protease hydrolysate (MW < 3 kDa) in Example 1. Figure 2 This is a schematic diagram illustrating the steps of ultrafiltration fractionation of castor silkworm pupa polypeptides and screening of α-glucosidase inhibitory peptides in Example 1. Figure 3 This is a two-dimensional schematic diagram illustrating the interaction between the three peptides screened in Example 1 and the non-specific site of α-glucosidase (PDB ID: 3WY1). Figure 1As shown, LC-MS / MS identified a total of 4,392 peptides. 52.89% of the peptides in the castor silkworm pupa trypsin hydrolysate had a molecular weight (MW) of <1 kDa, while only 8.52% had a MW of >1.5 kDa. The peptides contained all 20 protein amino acids, with hydrophobic amino acid content >45%, and their lengths were concentrated in the range of 4-10 amino acid residues. N-terminal amino acids and basic amino acids account for a relatively high proportion, while other types of amino acids are distributed relatively evenly. The top ten amino acids by quantity are: nonpolar aliphatic amino acids Ala, Leu, and Val, accounting for 6.12%, 16.23%, and 8.86%, respectively; aromatic amino acid Tyr, accounting for 4.83%; polar uncharged amino acids Ser and Thr, accounting for 5.56% and 11.36%, respectively; basic amino acid Lys, accounting for 5.49%; acidic amino acids Asp and Glu, accounting for 5.40% and 7.29%, respectively; and C-terminal basic amino acids are absolutely dominant, with arginine (Arg) and lysine (Lys), accounting for 12.89% and 53.83%, respectively.

[0052] (3) Determination of the inhibitory activity of α-glucosidase inhibitory peptides derived from castor silkworm pupae To verify the molecular docking results and elucidate the mechanism of action of the inhibitory peptides, three peptides, YYLER, TDPAF, and PPEF, were prepared via solid-phase synthesis. Their inhibitory activity against α-glucosidase was analyzed using three classical kinetic equations: Dixon, Cornish-Bowden, and Lineweaver-Burk. The results are as follows: Figure 4 As shown, castor silkworm pupa trypsin hydrolysate with MW < 3 kDa and the three synthetic peptides can effectively inhibit α-GC activity; the inhibition rates of YYLER, TDPAF, and PPEF all increased in a dose-dependent manner, with YYLER showing the best effect and an IC50 value of [missing value]. 50 The values ​​were 0.24 ± 0.05 mg / mL, 27.74 ± 1.38 mg / mL, and 23.20 ± 1.64 mg / mL, respectively. These results are compared with the binding energies obtained from molecular docking. Figure 3 The sorting is completely consistent.

[0053] Further, please refer to Figure 5Kinetic analysis was used to determine the inhibition pattern of YYLER: the Lineweaver-Burk equation curves intersect on the Y-axis, the Dixon curves intersect linearly at a point in the second quadrant, and the Cornish-Bowden curves are parallel. These characteristics indicate that as the inhibitory concentration increases, the maximum reaction rate (Vmax) remains constant, while the ratio of the Michaelis constant to the maximum reaction rate (Km / Vmax) increases, confirming that YYLER is a typical competitive inhibitor, directly competing with the substrate pNPG for the enzyme's active site. In contrast, TDPAF and PPEF exhibit mixed and non-competitive inhibitory activity, respectively. Therefore, kinetic analysis demonstrates that YYLER has stronger inhibitory activity compared to TDPAF and PPEF.

[0054] The specific information of the three peptide segments obtained in Example 1 is as follows: Peptide 1: The sequence is YYLER (Tyr-Tyr-Leu-Glu-Arg), containing 5 amino acid residues, with a molecular weight of 649.72 Da and a docking energy of -9.5 kcal / mol. Its IC50 value was determined by in vitro α-glucosidase inhibitory activity assay. 50 The value was 0.24 ± 0.05 mg / mL, which is a typical competitive inhibitor that can directly compete with the substrate pNPG for the active site of the enzyme. The peptide is non-sensitizing and non-toxic. It did not degrade after simulated gastrointestinal digestion and the gastrointestinal absorption rate reached 93%. It is a novel sequence that was first identified from castor silkworm pupae.

[0055] Peptide 2: The sequence is TDPAF (Thr-Asp-Pro-Ala-Phe), containing 5 amino acid residues, with a molecular weight of 524.56 Da, a docking energy of -9.0 kcal / mol, and an in vitro IC50 value. 50 The value was 27.74 ± 1.38 mg / mL, exhibiting a mixed inhibition pattern; it was non-sensitizing and non-toxic, retaining more than 91% of the active fragment after gastrointestinal digestion, with a gastrointestinal absorption rate of 88%, and was verified by databases as a novel peptide.

[0056] Peptide 3: The sequence is PPEF (Pro-Pro-Glu-Phe), containing 4 amino acid residues, with a molecular weight of 458.50 Da, a docking energy of -8.9 kcal / mol, and an in vitro IC50 value. 50 The value was 23.20 ± 1.64 mg / mL, indicating it is a non-competitive inhibitor; it is non-sensitizing and non-toxic, exhibits good gastrointestinal stability, and has a gastrointestinal absorption rate of 86%. It is a novel α-glucosidase inhibitor peptide that has not been previously reported.

[0057] The YYLER peptide exhibited the strongest α-glucosidase inhibitory activity, and the inhibitory effect of this peptide on α-glucosidase (IC50) was [missing value]. 50The concentration (0.24 ± 0.05 mg / mL) was superior to the inhibitory activity (IC50) of taro globulin against α-glucosidase reported in existing literature. 50 The inhibitory activity (IC50) of specific secondary metabolites isolated from *Balladina* species (such as *Balladina proflori* and *Balladina dentata*) against α-glucosidase was measured at 2.09 ± 0.19 mg / mL. 50 (0.296 mg / mL / 0.568 mg / mL) (Ma Erlan, Lin Ying, Tu Lian, et al. Extraction and purification of taro globulin and its inhibitory activity against α-amylase and α-glucosidase [J]. Science and Technology of Food Industry, 2021, 42(14):25-32. DOI: 10.13386 / j.issn1002-0306.2020100266; Deng Zhentao. Study on chemical components and bioactivity of four species of *Ballisneria* and *Diospyros kaki* [D]. Kunming: Kunming Institute of Botany, Chinese Academy of Sciences, 2025.).

[0058] (4) Thermodynamic characterization To further elucidate the thermodynamic binding mechanism of the YYLER peptide to α-glucosidase, isothermal titration calorimetry (ITC) was used for thermodynamic characterization. Figure 6 As shown, the original thermogram exhibits regular endothermic peaks (positive), and the fitted binding isotherm is in high agreement with the one-site binding model. The stoichiometric ratio n = 1.02 ± 0.03 indicates that YYLER and α-GC form a highly homogeneous complex in a 1:1 ratio. The dissociation constant KD = 1.66 ± 0.06 μmol·L⁻¹ -1 (310 K), corresponding to the binding constant Ka ≈ 6.0 × 10 5 L·mol -1 This indicates that YYLER has a high affinity for α-glucosidase.

[0059] The analysis of the above thermodynamic parameters shows that the enthalpy change of the combined reaction is ΔH = +2.8 ± 0.5 kcal・mol. -1 This indicates that the process is endothermic, and the enthalpy change is slightly unfavorable for bonding; however, the entropy change phenomenon -TΔS is -11.0 ± 1.2 kcal・mol -1 (ΔS>0), which makes a significant and favorable contribution to the binding free energy. Combining these two factors, we arrive at ΔG = -8.2 kcal·mol⁻¹. -1 (≈ -34.3 kJ·mol) -1The temperature (25 °C, 1 kcal = 4.184 kJ) indicates that the binding is spontaneous. Since |-TΔS| > |ΔH|, this binding process is a typical entropy-driven process. It is speculated that the hydrophobic effect (i.e., the release of water molecules from the binding interface) is the main driving force for complex formation. This high-affinity spontaneous binding characteristic supports YYLER's mechanism of action, which generates competitive inhibition by occupying the catalytic center.

[0060] (5) Effects of different proteases on screening To investigate the potential blood glucose control ability of castor silkworm pupae, single-enzyme hydrolysis control experiments were conducted using alkaline protease, papain, neutral protease, flavor protease, complex protease, pepsin, and trypsin (used in Example 1), respectively, in Comparative Examples 1-6. The α-glucosidase inhibitory activity of Examples 1 and Comparative Examples 1-6 was characterized. Using α-glucosidase inhibitory activity as the core evaluation index, the inhibition rate and half-inhibitory concentration (IC50) of each hydrolysate were measured. 50 The results are shown in Table 1. As can be seen from the data in Table 1, the hydrolysates obtained from different proteases significantly affected the inhibitory activity of α-glucosidase; among them, trypsin hydrolysate exhibited the strongest inhibitory activity, with an IC50 value of [missing value]. 50 The concentration was 6.5 ± 0.6 mg / mL, significantly lower than the hydrolysates of the other seven proteases in Comparative Examples 1-6 (p<0.05). Combined with enzymatic digestion characteristic analysis, trypsin specifically hydrolyzes the carboxyl-terminal peptide bonds of arginine and lysine, which highly matches the compositional characteristics of castor silkworm pupa protein, which is rich in basic and hydrophobic amino acids, enabling efficient release of short peptides with strong inhibitory activity. Further analysis of the activity results and enzyme compatibility in Table 1 demonstrates that trypsin is the optimal enzyme for the enzymatic digestion and preparation of α-glucosidase inhibitory peptides from castor silkworm pupae. Therefore, using trypsin is more advantageous for screening and obtaining the three peptides with stronger α-glucosidase inhibitory activity described in this invention.

[0061] Table 1 (6) Combining mechanism and structural representation To further elucidate the binding mechanism of YYLER with α-glucosidase, residue free energy decomposition, binding conformation analysis, and free energy landscape analysis were performed on the YYLER-α-glucosidase complex. Please refer to [link to relevant documentation]. Figure 7 ,in, Figure 7 A is a graph showing the results of residue free energy decomposition. Figure 7 B is a schematic diagram of the binding conformation of the key interacting residues. Figure 7 C represents the free energy landscape of the complex at equilibrium. (Example:) Figure 7As shown in Figure A, the residue free energy decomposition results indicate a significant electrostatic-desolvation compensation effect during the binding of YYLER with α-glucosidase. Charged residues such as Glu276, His239, and Arg312 exhibit strong electrostatic attraction to the ligand. Although subject to some polar desolvation penalty, their net free energy contribution remains beneficial and is the main thermodynamic driving force maintaining the stability of the complex. Figure 7 As shown in Figure B, Glu276 can form salt bridges and hydrogen bonds with YYLER, with an interaction distance of approximately 2.1 Å, which is beneficial for enhancing electrostatic interactions. Simultaneously, residues such as Phe300 stabilize YYLER within a hydrophobic subpocket through nonpolar interactions, indicating that electrostatic interactions and hydrophobic stacking synergistically endow the binding interface with high specificity and stability. Figure 7 As shown in C, during the 280–300 ns equilibrium phase, the free energy landscape exhibits a single and clear low-energy basin, indicating that the YYLER-α-glucosidase complex occupies a stable global lowest free energy conformation state, demonstrating that the complex has good conformational stability.

[0062] In summary, residue free energy decomposition, binding conformation analysis, and free energy landscape analysis collectively demonstrate that YYLER can achieve high-affinity binding by forming a stable and synergistic interaction network with multiple key residues within the α-glucosidase active pocket. Charged residues such as Glu276, His239, and Arg312 provide the main electrostatic driving force, while hydrophobic residues such as Phe300 further stabilize the ligand conformation. This synergistic effect provides a clear structural basis and mechanistic evidence for YYLER's α-glucosidase inhibitory activity.

[0063] (7) Screening and Mechanism Research Flowchart To systematically discover α-glucosidase inhibitory peptides derived from castor silkworm pupae, this invention constructs a comprehensive research system integrating enzymatic screening, de novo peptidomics identification, virtual screening, activity verification, and molecular mechanism analysis. Please refer to [link / reference]. Figure 8 , Figure 8 This is a flowchart illustrating the entire process of screening and mechanistic study of α-glucosidase inhibitory peptides derived from castor silkworm pupae. (See diagram below.) Figure 8 As shown, silkworm pupa protein was first prepared using castor silkworm pupae as raw material, and the optimal hydrolysis conditions were screened by different proteases. The results showed that the trypsin hydrolysate had the strongest α-glucosidase inhibitory activity, with an IC50 value of [missing value]. 50 The concentration was 6.5 ± 0.6 mg / mL, therefore it was selected as the target for subsequent active peptide screening. Subsequently, de novo sequencing yielded a total of 4392 peptide sequences. After safety screening, molecular docking, and stability evaluation, three superior candidate peptides were finally identified: YYLER, TDPAF, and PPEF. Among them, YYLER exhibited the strongest inhibitory activity, with an IC50 concentration of 6.5 ± 0.6 mg / mL.50 The concentration was 0.24 ± 0.05 mM, exhibiting competitive inhibition. Further thermodynamic analysis and molecular dynamics simulations showed that YYLER can stably bind to the active site of α-glucosidase and interact with key residues such as Glu276, His239, and Arg312. This binding process is spontaneous and exhibits an entropy-driven mode, indicating that YYLER has the potential to be used as a natural α-glucosidase inhibitory peptide in the development of functional foods for glycemic regulation.

[0064] In summary, this invention establishes a systematic screening and mechanistic study process for α-glucosidase inhibitory peptides derived from castor silkworm pupae, achieving a complete process from by-product resource development, determination of optimal enzymatic hydrolysis conditions, identification of active peptide sequences, virtual selection of candidate peptides, to in vitro functional verification and elucidation of the mechanism of action. The results demonstrate that YYLER is a novel natural α-glucosidase competitive inhibitory peptide derived from castor silkworm pupae, possessing good inhibitory activity, safety, stability, and application potential. It can provide a theoretical basis and technical support for the high-value utilization of castor silkworm pupae by-products and the development of natural glycemic regulatory functional foods.

[0065] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0066] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A castor silkworm pupa hydrolysate, characterized in that, The castor silkworm pupa hydrolysate contains at least one α-glucosidase inhibitory peptide with the amino acid sequence Tyr-Tyr-Leu-Glu-Arg, Thr-Asp-Pro-Ala-Phe, or Pro-Pro-Glu-Phe.

2. The castor silkworm pupa hydrolysate according to claim 1, characterized in that, The amino acid sequence of the α-glucosidase inhibitory peptide contained in the castor silkworm pupa hydrolysate is Tyr-Tyr-Leu-Glu-Arg.

3. A method for screening active peptides from castor silkworm pupa hydrolysate as described in any one of claims 1 to 2, characterized in that, Includes the following steps: S1, defatted castor silkworm pupa powder: freeze-dried castor silkworm pupa powder is mixed with n-hexane at a ratio of 1:(4-6) (g / mL) at 20-30℃, then centrifuged several times at 4℃ and the supernatant is discarded. After drying, defatted castor silkworm pupa powder is obtained. S2, Preparation of castor silkworm pupa protein: The defatted castor silkworm pupa powder was mixed with distilled water, and the pH was adjusted to 10.5-11.5 with NaOH. Then the pH was adjusted to 4.0-5.0 with HCl and stirred evenly. The mixture was centrifuged at 4°C, and the resulting precipitate was dialyzed and freeze-dried to obtain castor silkworm pupa protein. S3, Castor silkworm pupa protein hydrolysis: The castor silkworm pupa protein and distilled water were mixed evenly at a ratio of 1:(8-12) (g / mL). The pH of the solution was adjusted to 7.0-8.0 with NaOH. 600 U / mL trypsin was added at 45-50℃ and the reaction was carried out with constant temperature shaking for 3-5 hours. The reaction was then terminated by heating in a water bath at 95℃ for 15 minutes. The mixture was centrifuged at 4℃, the supernatant was separated and freeze-dried to obtain castor silkworm pupa trypsin hydrolysate. S4, Ultrafiltration fractionation and component identification of castor silkworm pupa peptides: Castor silkworm pupa protein hydrolysate was reconstituted in distilled water at a ratio of 1:(10-20) (g / mL), and fractionated using a centrifugal ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected and freeze-dried to obtain castor silkworm pupa-derived active peptide components with a molecular weight less than 3 kDa. The castor silkworm pupa-derived active peptide components were pretreated by dissolution, reductive alkylation, and SP2 desalting. The molecular weight distribution, amino acid composition, peptide chain length, and N / C-terminal amino acid types of the castor silkworm pupa-derived active peptide components were detected and identified using liquid chromatography-mass spectrometry. S5, Screening of α-glucosidase inhibitory peptides: First, the active peptide components derived from castor silkworm pupae were subjected to pLM4Alg and ToxinPred 3.0 platform to remove sensitizing / toxic sequences, and the remaining candidate peptides were used for molecular docking. The ligand molecular structure was constructed using ChimeraX 1.10 and saved in PDBQT format. Molecular docking analysis was performed using AutoDock 4.2 to screen for high-affinity candidate peptides. The high-affinity candidate peptides were simulated in the gastrointestinal digestive environment using BIOPEP-UWM to analyze the undegraded peptides or newly generated active fragments after enzymatic hydrolysis, and the gastrointestinal absorption rate of the peptides was predicted using SwissADME. The candidate peptides were ranked based on three priority indicators: absolute binding energy, number of anti-digestive fragments, and absorption potential. The undegraded candidate peptides obtained were the active peptides of the castor silkworm pupae hydrolysate.

4. The method for screening active peptides from castor silkworm pupa hydrolysate according to claim 3, characterized in that, The specific steps of S1 are as follows: Fresh castor silkworm pupae were washed, killed with liquid nitrogen, freeze-dried, and ground through an 80-mesh sieve. The freeze-dried castor silkworm pupae powder was then mixed with n-hexane at a ratio of 1:5 (g / mL), stirred at 400 rpm for 4 h at 25°C, centrifuged at 4000 rpm for 10 min at 4°C and the supernatant was discarded. The centrifugation operation was repeated three times, and the powder was dried with nitrogen to obtain defatted castor silkworm pupae powder.

5. The method for screening active peptides from castor silkworm pupa hydrolysate according to claim 3, characterized in that, The specific steps in S2 are as follows: The defatted castor silkworm pupa powder was mixed with distilled water at a ratio of 1:10 (g / mL) until homogeneous. The pH was adjusted to 11.0 with 1 mol / L NaOH. After 6 h, the pH was adjusted to 4.5 with 1 mol / L HCl and stirred for 4 h. The mixture was then centrifuged at 10,000 rpm for 15 min at 4 °C. The precipitate was dialyzed and freeze-dried to obtain the castor silkworm pupa protein.

6. The method for screening active peptides from castor silkworm pupa hydrolysate according to claim 3, characterized in that, The specific steps of S3 are as follows: The castor silkworm pupa protein was mixed with distilled water at a ratio of 1:10 (g / mL) until homogeneous. The pH of the solution was adjusted to 7.5 with 1 mol / L NaOH. 600 U / mL trypsin was added at 47°C and the mixture was subjected to constant temperature shaking reaction for 4 h. The reaction was then terminated by heating in a 95°C water bath for 15 min. The supernatant was separated by centrifugation at 10,000 rpm for 15 min at 4°C. The supernatant was then freeze-dried to obtain the castor silkworm pupa trypsin hydrolysate.

7. The method for screening active peptides from castor silkworm pupa hydrolysate according to claim 3, characterized in that, The specific steps in S4 are as follows: The castor silkworm pupa protein hydrolysate was reconstituted in distilled water at a ratio of 1:15 (g / mL). The supernatant after reconstitution was taken and added to a 3 kDa ultrafiltration tube. The mixture was centrifuged at 10,000 rpm for 15 min at 4°C. Peptide fractions with a molecular weight of less than 3 kDa were collected from the permeate. The permeate was collected and freeze-dried to obtain active peptide fractions derived from castor silkworm pupae with a molecular weight of less than 3 kDa. The active peptide components derived from castor silkworm pupae were pretreated by dissolution, reductive alkylation, and SP2 desalting, and then detected using an Easy-nLC 1200 / QExactive liquid chromatography-mass spectrometry system. Liquid chromatography conditions: pre-column 150 μm id × 50 mg / mL, analytical column 150 μm id × 170 mg / mL, mobile phase A 0.1% FA aqueous solution, mobile phase B 0.1% FA-80% acetonitrile solution, flow rate 600 nL / min, analysis time 66 min. Mass spectrometry conditions: acquisition mode DDA, primary mass spectrometry resolution 70000, AGC target 3e6, scan range 100-1500 m / z, secondary mass spectrometry resolution 17500, AGC target 1e5, NCE 28. The raw mass spectrometry data were analyzed using PEAKS De novo sequencing technology to identify and obtain the peptide sequence information of the active peptide components derived from castor silkworm pupae, including molecular weight distribution, amino acid composition, peptide chain length, and N / C-terminal amino acid types. The search parameters were as follows: fixed modification was Carbamidomethyl (C), variable modification was Oxidation (M) or Acetyl (Peptide N-term), enzyme type was Non-specific, precursor ion mass tolerance was 20 ppm, and fragment ion mass tolerance was 0.02 Da.

8. The method for screening active peptides from castor silkworm pupa hydrolysate according to claim 3, characterized in that, The specific steps in S5 are as follows: After removing sensitizing / toxic sequences from the castor silkworm pupa-derived active peptide components using the pLM4Alg and ToxinPred 3.0 platforms, the remaining candidate peptides were used for molecular docking. The ligand molecular structure was constructed using ChimeraX 1.10 and saved in PDBQT format. Molecular docking analysis was performed using AutoDock 4.2 to screen for candidate peptides with high affinity. The specific parameters for the molecular docking analysis were as follows: the grid center coordinates (center_x, center_y, center_z) were set to [3.250, -8.25, -2.556], the grid center was set at the enzyme active site (60×60×60 Å, spacing 0.375 Å), the genetic algorithm was run 10 times, the maximum energy evaluation was 2.5 million times, and the binding free energy ΔG < -7.5 kcal / mol was used as the threshold. The high-affinity candidate peptides were simulated using BIOPEP-UWM to analyze the undegraded peptides or newly generated active fragments after enzymatic hydrolysis, and the gastrointestinal absorption rate of the peptides was predicted using SwissADME. The conditions for simulating the gastrointestinal digestive environment were: pepsin pH 3.0, trypsin / chymotrypsin pH 7.6, and 37°C. The candidate peptides were ranked based on three priority indicators: absolute binding energy, number of anti-digestion fragments, and absorption potential. The undegraded candidate peptides obtained were the active peptides of the castor silkworm pupa hydrolysate.

9. An α-glucosidase inhibitor, characterized in that, The active peptides include those obtained by screening the castor silkworm pupa hydrolysate as described in any one of claims 1 to 2 or by screening the castor silkworm pupa hydrolysate as described in any one of claims 3 to 8.

10. The use of the castor silkworm pupa hydrolysate as described in any one of claims 1 to 2 or the α-glucosidase inhibitor as described in claim 9 in the preparation of hypoglycemic drugs.

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