Hypoglycemic peptide, synthesis method and application thereof
By extracting and purifying peptides from Hericium erinaceus fruiting bodies, a hypoglycemic peptide with the amino acid sequence SLAALSTAKSTLAAGQGGPEAAQK was synthesized, solving the problems of the lack of research on Hericium erinaceus fruiting bodies and the insignificant effects of edible fungus extracts in the existing technology, and achieving a significant hypoglycemic effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI ACAD OF AGRI SCI
- Filing Date
- 2025-06-30
- Publication Date
- 2026-04-10
AI Technical Summary
In the existing technology, there are no reports on the research of hypoglycemic peptides derived from Hericium erinaceus fruiting bodies, and the existing edible fungi extracts have no significant effect on the inhibition rate of α-amylase, making it difficult to effectively control postprandial hyperglycemia.
Peptides were extracted and purified from the fruiting bodies of Hericium erinaceus. A hypoglycemic peptide with the amino acid sequence SLAALSTAKSTLAAGQGGPEAAQK was synthesized using a solid-phase synthesis method. After purification by multi-step chromatography and dialysis, it was identified as the main active ingredient and applied to the preparation of drugs that inhibit the activity of α-amylase and α-glucosidase.
The synthesized hypoglycemic peptides showed an inhibition rate of 68.14%±0.27% against α-amylase and 68.14%±0.34% against α-glucosidase, significantly improving the hypoglycemic effect and demonstrating significant hypoglycemic activity and safety.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological extracts, and relates to a hypoglycemic peptide as well as a synthesis method and application thereof. BACKGROUND
[0002] Diabetes has become the third chronic non-communicable disease after cardiovascular and cerebrovascular diseases and malignant tumors. Hyperglycemia is the most important feature of diabetes, and controlling postprandial hyperglycemia is an effective and attractive target for treating diabetes. In daily life, carbohydrates mainly exist in the form of polysaccharides, which makes them unable to be directly absorbed and utilized by the human body. In the gastrointestinal tract, alpha-glucosidase and alpha-amylase are responsible for the decomposition of oligosaccharides and disaccharides into monosaccharides for absorption by the human body. Therefore, inhibiting alpha-glucosidase and alpha-amylase can delay glucose absorption in the body, thereby reducing postprandial blood glucose levels. According to research, hypoglycemic peptides extracted from natural products have the activity of inhibiting alpha-amylase and alpha-glucosidase, and have less toxicity and side effects on the human body compared with chemically synthesized drugs.
[0003] A large number of studies have shown that edible fungi are rich in various active substances with hypoglycemic effects, such as proteins, peptides, polysaccharides, and terpenoids. At the same time, compared with animals and plants, edible fungi have a short growth cycle and are easy to cultivate, making them a high-quality material for developing hypoglycemic drugs. Liu Bing et al. studied the isolation and purification of extracellular polysaccharides from mycelium of Morchella esculenta and their hypoglycemic and antioxidant activities. The inhibition rates of extracellular polysaccharides MEP-H and MEP-N from Morchella esculenta on alpha-glucosidase were 74.93%±2.72% and 69.48%±2.97%, respectively, and the highest inhibition rates on alpha-amylase were 8.06%±1.93% and 11.08%±1.05%, respectively, showing certain hypoglycemic activity, but the inhibition rate on alpha-amylase was not significantly effective (Liu Bing, Li Pei, Zeng Xinyu, et al. Isolation and purification of extracellular polysaccharides from mycelium of Morchella esculenta and their hypoglycemic and antioxidant activities [J]. Journal of Food Safety and Quality Inspection, 2025, 16(05): 86-93.).
[0004] Hericium erinaceus contains various active ingredients, especially rich proteins. So far, there has been no related report on hypoglycemic peptides derived from the fruiting bodies of Hericium erinaceus. SUMMARY
[0005] The application provides a hypoglycemic peptide as well as a synthesis method and application thereof.
[0006] The hypoglycemic peptide has an amino acid sequence of SLAALSTAKSTLAAGQGGPEAAQK, that is, Ser-Leu-Ala-Ala-Leu-Ser-Thr-Ala-Lys-Ser-Thr-Leu-Ala-Ala-Gly-Gln-Gly-Gly-Pro-Glu-Ala-Ala-Gln-Lys, as shown in SEQ ID NO. 1.
[0007] The hypoglycemic peptide can be synthesized by a solid-phase synthesis method.
[0008] The application provides application of the hypoglycemic peptide in preparation of a medicine for inhibiting activities of alpha-amylase and alpha-glucosidase.
[0009] Further, the application provides application of the hypoglycemic peptide in preparation of a hypoglycemic medicine.
[0010] Compared with the prior art, the application has the following advantages:
[0011] The polypeptide is extracted and purified from a Hericium erinaceus fruiting body for the first time, and a plurality of hypoglycemic peptides with good blood sugar lowering capacity are found, and the amino acid sequence of one of the hypoglycemic peptides is determined as SLAALSTAKSTLAAGQGGPEAAQK by LC-MS / MS, the alpha-amylase inhibition rate is 68.14%±0.27%, and the alpha-glucosidase inhibition rate is 68.14%±0.34%, the effect is significant, and the polypeptide can be used as a natural hypoglycemic peptide and has a potential application prospect in the field of preparation of a blood sugar lowering medicine. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 C1, C2 and C3 are three polypeptides with different molecular weights, and the alpha-glucosidase and alpha-amylase inhibition rates are shown.
[0013] Figure 2 Q1, Q2 and Q3 are products separated by an anion column.
[0014] Figure 3 q1, q2 and q3 are products separated by an anion column.
[0015] Figure 4 The alpha-glucosidase inhibition rates of Q1, Q2 and Q3 separated by an anion column are shown.
[0016] Figure 5 The alpha-glucosidase inhibition rates of q1, q2 and q3 separated by an anion column are shown.
[0017] Figure 6To separate products G1, G2, G3, G4 by gel column.
[0018] Figure 7 To separate the α-glucosidase inhibition rate of products G1, G2, G3, G4.
[0019] Figure 8 TIC map of G2.
[0020] Figure 9 Secondary mass spectrum of active polypeptide.
[0021] Figure 10 Prediction of active polypeptide structure. DETAILED DESCRIPTION
[0022] The application will be further described below in conjunction with specific examples and drawings.
[0023] In the following examples, the determination of α-amylase inhibition rate refers to the method in (Sunna. Preparation and identification of hypoglycemic bioactive peptides in camel milk proteins [D]. Hohhot: Inner Mongolia Agricultural University, 2020.) and the determination of α-glucosidase inhibition rate refers to the method in (Zhao Hongxing. Preparation of hypoglycemic active peptides, purification and identification of α-glucosidase activity peptides [D]. Harbin: Harbin Institute of Technology, 2018.).
[0024] Example 1
[0025] (1) Take fresh Hericium erinaceus fruiting bodies, wash and cut into small pieces for liquid nitrogen quick freezing, then freeze-dry using a freeze-drying machine, then use a pulverizer to pulverize the freeze-dried fruiting bodies into powder and pass through a 60-mesh sieve. Mix the powder and ultrapure water according to a mass ratio of 1:10, dissolve by ultrasonic, and let it stand for 4 h. Centrifuge the solution at 4 ℃ and 8000 r / min for 20 min, then take out the upper liquid, add ammonium sulfate to a final concentration of 80% saturation (add 561 g of solid ammonium sulfate per liter of solution), ultrasonic to dissolve, and stand for 12 h. Then centrifuge the solution at 4 ℃ and 8 000 r / min for 20 min to obtain the precipitate. Dissolve the precipitate in ultrapure water, then put it into a 10 kDa dialysis bag and dialyze in a 4 ℃ water solution environment for 48 h. After completion, freeze-dry the powder to obtain Hericium erinaceus protein.
[0026] (2) The Hericium erinaceus protein was dissolved in ultrapure water, and 1 mol / L sodium hydroxide and hydrochloric acid were used to adjust the environment to the suitable conditions (pH 9, 55°C) for alkaline protease. After the environment reached a stable state, 4% of the substrate mass of alkaline protease was added, and the enzymolysis was performed for 4 hours. Then, the reaction was terminated by water bath at 90°C for 15 minutes. Finally, the supernatant was obtained by centrifugation at 4°C and 8,000 r / min for 15 min, and the enzymolysis product was obtained by freeze-drying. The active peptide obtained after enzymolysis was dissolved and filtered through a 0.22 μm filter membrane. Three polypeptide components with different molecular weights were obtained by centrifugation at 4,000 r / min for 60 min using 10 kDa and 3 kDa ultrafiltration tubes, and were named C1, C2, and C3, respectively. The inhibitory rates of the three polypeptide components with different molecular weights on α-glucosidase and α-amylase were tested. The results are shown in Figure 1 As can be seen from the figure, the polypeptide component with the optimal inhibitory rates on α-amylase and α-glucosidase is C1.
[0027] (3) C1 was purified by Q Sepharose FF anion column through first step gradient purification (eluent is NaCl (2 M pH 7.5) B solution). As shown in FIG. 2, three elution peaks were produced, which were Q1, Q2, and Q3, respectively. The hypoglycemic activity of the three elution peaks was evaluated by the inhibitory rate on α-glucosidase, and the results are shown in FIG. 4. Among the three components, Q2 showed better activity, and the inhibitory rate on α-glucosidase was 0.4974±0.0166. According to the gradient at which Q2 appeared, which was 25% B (the proportion of Buffer B was 25%), the elution conditions were optimized again, and C1 was eluted by Q Sepharose FF anion column at 0-25% B linearly, and 20 column volumes were eluted, as shown in Figure 3 Three elution peaks were produced, which were q1, q2, and q3, respectively. The hypoglycemic activity of the three elution peaks was evaluated by the inhibitory rate on α-glucosidase, and the results are shown in Figure 5 As can be seen from the figure, q2 showed better activity, and the inhibitory rate on α-glucosidase was 62.95±0.13%.
[0028] q2 separated from the anion column was further purified by Superdex 30 Increase 10 / 300 GL gel column and detected by 220 nm absorbance, and four different components were successfully separated, and the results are shown in Figure 6The four components obtained are named G1, G2, G3, and G4 in turn according to the elution order. The gel column is used to separate and purify the components according to the molecular weight, and the inhibitory rates of G1, G2, G3, and G4 on α-glucosidase are determined respectively, and the results are shown in Table 1. Figure 7 The hypoglycemic activity of G2 is significantly higher than that of the other three components, and the inhibitory rate on α-glucosidase is as high as 83.24±0.37%. At the same time, compared with the hypoglycemic activity of q2 after ion column purification, the hypoglycemic activity has been improved to a certain extent, which indicates that the hypoglycemic peptide of Hericium erinaceus has been further purified, and its hypoglycemic activity has been improved, and the hypoglycemic peptide of Hericium erinaceus is mainly concentrated in G2.
[0029] (4) The amino acid sequence of the hypoglycemic peptide at G2 of Hericium erinaceus is determined by LC-MS / MS. Figure 8 The TIC spectrum of G2 of Hericium erinaceus is obtained. The 33 sequences are uploaded to PeptideRanker for activity prediction. PeptideRanker gives scores of 34 peptides in G2, one of which has a score higher than 0.5, indicating that the peptide shows a high possibility of biological activity. The amino acid sequence of the hypoglycemic active peptide is SLAALSTAKSTLAAGQGGPEAAQK, as shown in SEQ ID NO. 1, and its mass spectrum is shown in FIG. 5. Figure 9 The molecular weight is 2230.16 Da.
[0030] (5) The performance of the SLAALSTAKSTLAAGQGGPEAAQK peptide is analyzed by bioinformatics. The ProParam online software predicts the physicochemical properties of the peptide: the theoretical isoelectric point is 8.31; it has one positively charged amino acid residue and one negatively charged amino acid residue; the stability is 21.97, which has good stability; the predicted half-life in mammalian reticulocytes is 1.9 h, the half-life in yeast is 20 h, and the half-life in E. coli is greater than 10 h; it is a non-hydrophilic polypeptide.
[0031] The safety of the obtained hypoglycemic peptide of Hericium erinaceus is tested by Toixinpred software, and its toxicity and allergenicity are predicted. The results show that it is non-toxic and non-allergic, and has high safety.
[0032] (6) SLAALSTAKSTLAAGQGGPEAAQK peptide was obtained by solid-phase synthesis method in Shenguo Bioengineering Co., Ltd., and the hypoglycemic activity of SLAALSTAKSTLAAGQGGPEAAQK peptide was determined. The α-amylase inhibition rate was 68.14%±0.27%, and the α-glucosidase inhibition rate was 68.14%±0.34%. PepDraw was used to predict the structure of the obtained Hericium erinaceus hypoglycemic peptide, and ChemDraw was used to draw its 2D structure. The results are shown in Figure 10 .
Claims
1. A blood glucose lowering peptide, characterized in that, The amino acid sequence is SLAALSTAKSTLAAGQGGPEAAQK.
2. The method of synthesizing a blood glucose-lowering peptide according to claim 1, characterized in that, The method of solid phase synthesis is used.
3. Use of the hypoglycemic peptide of claim 1 in the preparation of a hypoglycemic medicament.