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CN-122011089-A - Lumbricus-derived alpha-glucosidase inhibitory peptide and application thereof

CN122011089ACN 122011089 ACN122011089 ACN 122011089ACN-122011089-A

Abstract

The invention discloses an alpha-glucosidase inhibitory peptide derived from earthworm and application thereof, wherein the alpha-glucosidase inhibitory peptide is prepared by taking earthworm (Eisenia foetida) as a raw material, carrying out autolysis pretreatment, carrying out secondary enzymolysis by adopting flavourzyme, combining ultrafiltration separation (selecting <3kDa component), LC-MS/MS peptide sequence identification, and screening by combining PEPTIDERANKER, AUTODOCKVINA and molecular docking software, wherein the amino acid sequences are Arg-Gly-Phe (RGF) and Phe-Leu-Pro (FLP). The IC 50 values of RGF and FLP are respectively 0.599mg/mL and 1.344mg/mL by taking the inhibition activity of alpha-glucosidase as an index. The alpha-glucosidase inhibitory peptide provided by the invention can be used as an auxiliary hypoglycemic active ingredient and can be applied to functional products such as food, special medical food, medicines and the like.

Inventors

  • ZHOU FEIYAN
  • LI DAN
  • WU HUI
  • CHEN MENGZHANG
  • LAI FURAO

Assignees

  • 广州白云山维一实业股份有限公司

Dates

Publication Date
20260512
Application Date
20260122

Claims (10)

  1. 1. The alpha-glucosidase inhibitory peptide is characterized in that the amino acid sequence of the alpha-glucosidase inhibitory peptide is Arg-Gly-Phe or Phe-Leu-Pro, the Arg-Gly-Phe is abbreviated as RGF, and the Phe-Leu-Pro is abbreviated as FLP.
  2. 2. The earthworm-derived alpha-glucosidase inhibitory peptide as in claim 1, wherein the amino acid sequence is selected from earthworm protein hydrolysate, EPH, by a virtual screening technique.
  3. 3. The earthworm-derived alpha-glucosidase inhibitory peptide as in claim 2, wherein the earthworm protein hydrolysate EPH is prepared by the steps of: (1) Raw material treatment, namely soaking fresh earthworms in physiological saline water for spitting sand, washing with clear water after finishing, and draining water. Homogenizing fresh Lumbricus with beater, packaging, and storing at low temperature; (2) Autolysis, namely adding water into earthworm homogenate, stirring for pre-enzymolysis, stopping the reaction in a boiling water bath after the pre-enzymolysis is finished, and cooling; (3) And (3) secondary enzymolysis, namely adding flavourzyme into the system after autolysis enzymolysis is finished, regulating the pH and the temperature of the enzymolysis system, carrying out secondary enzymolysis under the stirring condition, terminating the reaction in a boiling water bath after the enzymolysis reaction is finished, cooling, and centrifugally collecting supernatant to obtain the earthworm protein hydrolysate EPH.
  4. 4. An alpha-glucosidase inhibitory peptide derived from earthworm as claimed in claim 3, wherein in step (2), the mass ratio of the earthworm homogenate to water is 1:1-1:4.
  5. 5. An alpha-glucosidase inhibitory peptide derived from earthworm as claimed in claim 3, wherein in step (3), the pH and temperature of the enzymolysis system are adjusted to 7.5-10.5, and the temperature is 45-60 ℃.
  6. 6. An alpha-glucosidase inhibitory peptide derived from earthworm as claimed in claim 3, wherein in step (3), the flavourzyme is added in an amount of 3000 u/g-7000 u/g in terms of protein content, and the enzymolysis time is 2-5 hours.
  7. 7. The earthworm derived alpha-glucosidase inhibitory peptide as in claim 2, wherein the virtual screening technique comprises the steps of: (1) Centrifuging Lumbricus protein hydrolysate EPH, intercepting different molecular weight components by ultrafiltration membrane to verify alpha-glucosidase activity, and freeze-drying the component with the best inhibition effect on alpha-glucosidase activity; (2) Identifying peptide sequences of components with the best alpha-glucosidase activity inhibition effect, and obtaining the amino acid sequence of the potential active peptide through database retrieval and analysis; (3) Screening 10lgP >20 peptide fragments, carrying out activity scoring on the obtained potential active peptide sequences, screening peptide sequences with scores of more than 0.9, and then screening the potential active peptides according to the amino acid composition characteristics of the alpha-glucosidase inhibitory peptide; (4) Performing docking analysis on candidate active peptides and alpha-glucosidase protein with PDB ID of 3A4J, calculating docking scores Vina score, and screening Vina score < -8.9 peptide sequences; (5) And (3) carrying out in-vitro alpha-glucosidase inhibitory activity verification on the peptide sequences obtained by screening, and measuring half inhibition concentration.
  8. 8. The earthworm-derived α -glucosidase inhibitory peptide of claim 1, wherein the earthworm-derived α -glucosidase inhibitory peptide is capable of being synthesized according to the amino acid sequence, the synthesis comprising synthesis using a solid phase synthesis method, wherein the solid phase synthesis method comprises Fmoc-SPPS mode, BOC-SPPS mode, and fragment condensation ligation mode.
  9. 9. Use of the earthworm-derived alpha-glucosidase inhibitory peptide as in claim 1, for the preparation of a product for assisting in lowering blood sugar.
  10. 10. The use according to claim 9, wherein the preparation of the supplemental hypoglycemic product comprises the use of an alpha-glucosidase inhibitory peptide as an active ingredient in the preparation of a supplemental hypoglycemic food, a special medical use formula or a pharmaceutical product.

Description

Lumbricus-derived alpha-glucosidase inhibitory peptide and application thereof Technical Field The invention relates to bioactive peptide and application, in particular to alpha-glucosidase inhibitory peptide derived from earthworm and application thereof. Background Type 2 diabetes is a metabolic disease characterized by hyperglycemia, which is caused by insulin action disorders, often accompanied by metabolic disorders such as sugar, protein, and fat. Long-term hyperglycemia and metabolic disorders in the body can further induce systemic tissue organ lesions, including kidney, cardiovascular, ocular, nervous system lesions, skin infections, and the like. Therefore, lowering blood glucose concentration in humans has a positive effect on the prevention and treatment of diabetes in a scientific range. Inhibition of alpha-glucosidase activity is considered to be a currently effective method of treating type 2 diabetes. More α -glucosidase inhibitors are used clinically as chemical synthesis drugs. Although these effects are excellent, they cause adverse reactions and side effects such as liver injury, myocardial infarction, edema, anemia, etc. after long-term administration. Thus, natural active ingredients that are safe and nontoxic to screen and have an alpha-glucosidase inhibitory effect have been of interest to researchers. Food-borne alpha-glucosidase inhibitory peptides have attracted considerable attention from scientific researchers due to their nature, safety, high efficiency, etc., for example, tripeptides GEY (IC 50 is 2.70 mg/mL), GYG (IC 50 is 1.50 mg/mL) isolated from silkworm cocoon hydrolysates, and peptide-fragment PFP (IC 50 is 3.10 mg/mL) obtained from Aspergillus oryzae fermentation products, all of which exhibit good alpha-glucosidase inhibitory activity （Lee,H.J.,Lee,H.S.,Choi,J.W.,Ra,K.S.,Kim,J.M.,&Suh,H.J.(2011).Novel tripeptides with alpha-glucosidase inhibitory activity isolated from silk cocoon hydrolysate.Journal of Agricultural and Food Chemistry,59 (21),1152211525;Kang,M.G.,Yi,S.H.,Lee,J.S.(2013).Production and characterization of a new α-glucosidase inhibitory peptide from Aspergillusoryzae N1591,Mycobiology,41:3,149154). However, the traditional food-borne polypeptide preparation process has significant limitations that a series of complex steps such as protein extraction, enzymolysis, hydrolysate separation and purification, active polypeptide characterization and identification are required, and the traditional food-borne polypeptide preparation process is complex in operation, long in time consumption and high in cost, so that practical application transformation is limited. The advent of virtual screening techniques has provided new ideas for the discovery of active polypeptides. Virtual screening is a method for rapid screening of active molecules of interest from complex databases by assessing the interaction between ligands and receptors. This method has been widely used to screen and discover naturally active molecules. Earthworm (Eisenia foetida) is used as a traditional animal-derived traditional Chinese medicine in China, has a long application history, has a protein content of 53.5% -63.1%, and has been proved to contain various components with biological activities such as anticoagulation, thrombolysis, immunoregulation and the like. Lumbricus protein is listed as a new resource food in 2009 in China, and policy support is provided for application of the Lumbricus protein in the fields of functional foods and medicines. The rich protein resources of the earthworms provide a good material basis for the excavation of active peptides, but the research on alpha-glucosidase inhibitory peptides of earthworm sources is still deficient at present, although the prior art has relevant reports on the preparation of the active peptides by animal-derived protein hydrolysates, the optimization of enzymolysis technology is focused, specific earthworm peptide sequences with alpha-glucosidase inhibitory activity are not clearly obtained, and meanwhile, the method for targeted screening of the alpha-glucosidase inhibitory peptides of earthworm sources by utilizing a virtual screening technology is not reported in the related research. Disclosure of Invention The invention aims to provide an alpha-glucosidase inhibitory peptide derived from earthworm and application thereof, aiming at overcoming the defects in the prior art, and making up the blank of a specific alpha-glucosidase inhibitory peptide sequence in earthworm. The invention provides an alpha-glucosidase inhibitory peptide derived from earthworm, wherein the amino acid sequence of the alpha-glucosidase inhibitory peptide is Arg-Gly-Phe or Phe-Leu-Pro, arg-Gly-Phe is abbreviated as RGF, and Phe-Leu-Pro is abbreviated as FLP. Preferably, the amino acid sequence is selected from the earthworm protein hydrolysate EPH by a virtual screening technique. Further preferably, the earthworm protein hydrolysate EPH is prepared by the f