Two polypeptides with alpha-glucosidase inhibitory activity and use thereof
By identifying and synthesizing the peptides EGEPKLP and TPElkL from shiitake mushrooms, the adverse reaction problems of existing chemically synthesized α-glucosidase inhibitors have been solved, providing highly active and safe natural α-glucosidase inhibitors for controlling postprandial blood glucose.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
- Filing Date
- 2023-06-22
- Publication Date
- 2026-07-21
Smart Images
Figure CN116751251B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and specifically relates to two polypeptides with α-glucosidase inhibitory activity and their applications. Background Technology
[0002] Diabetes mellitus is a widespread metabolic disorder. According to data from the International Diabetes Federation, 537 million adults worldwide had diabetes in 2021, with 90% of these cases being type 2 diabetes. Hyperglycemia is the main characteristic of type 2 diabetes. Long-term hyperglycemia can lead to chronic damage and dysfunction of various tissues, causing complications such as retinal damage and chronic kidney failure. Therefore, controlling blood sugar, especially postprandial blood sugar, is an effective treatment for type 2 diabetes, and delaying carbohydrate metabolism is a key measure for controlling postprandial blood sugar. Alpha-glucosidase, located in the epithelial mucosa of the small intestine, is a key enzyme involved in carbohydrate metabolism. Alpha-glucosidase inhibitors can control the rate of blood sugar production by inhibiting the activity of alpha-glucosidase and thus slowing down carbohydrate metabolism. Currently, alpha-glucosidase inhibitors are widely used in diabetes treatment, such as acarbose, voglibose, and miglitol. However, long-term use of these chemically synthesized drugs can lead to adverse reactions. Therefore, the search for alpha-glucosidase inhibitors from natural foods as a complementary therapy for type 2 diabetes has received widespread attention.
[0003] Bioactive peptides are compounds consisting of two or more amino acids linked by peptide bonds. They play important physiological roles in the human body and are characterized by good solubility, high activity, easy digestion and absorption, and safety and non-toxicity, thus attracting increasing attention. Recent studies have also found that hydrolyzed peptides from ham, soybeans, amaranth, eggs, and quinoa proteins possess α-glucosidase inhibitory activity. Currently, various α-glucosidase inhibitory peptides have been identified from hydrolyzed proteins such as dried ham, buffalo milk, chickpeas, and black tea, including the IC50 of AEEEYPDL, LGVGG, and GGLGP from dried ham. 50 The concentrations were 5.58 mM (5.38 mg / mL), 6.36 mM (2.55 mg / mL), and 8.71 mM (3.48 mg / mL), respectively; the IC50 values of YLGYLEQLLR, TKVIPYVRYL, and RNAVPITPTLNR in buffalo milk protein were... 50 The concentrations were 470.5 μM (0.596 mg / mL), 498.0 μM (0.623 mg / mL), and 503.6 μM (0.681 mg / mL), respectively; the IC50 values of SPGAGKG and GLAR in chickpea protein were... 50 The IC50 values for CGKKFVR and AVPANLVDLNVPALLK in black tea protein were 1.78 mg / mL and 8.74 mg / mL, respectively. 50The concentrations were 0.52 mg / mL and 1.03 mg / mL, respectively. Note: The above IC50 values... 50 This refers to the inhibitory activity of α-glucosidase.
[0004] The invention "Tripeptide CGP with hypoglycemic function and its uses" (2017103998437) states that the α-glucosidase inhibitory activity of tripeptide CGP at 5.0 mg / mL is 35.9%.
[0005] The invention "Tripeptide SPF with hypoglycemic function and its uses" (2017104006528) states that the α-glucosidase inhibitory activity of tripeptide SPF at 5.0 mg / mL is 30.5%. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to provide two new polypeptides with α-glucosidase inhibitory activity and their uses.
[0007] To address the aforementioned technical problems, this invention provides the following two polypeptides with α-glucosidase inhibitory activity:
[0008] The polypeptide EGEPKLP has the following amino acid sequence: Glu-Gly-Glu-Pro-Lys-Leu-Pro.
[0009] The polypeptide TPELKL has the following amino acid sequence: Thr-Pro-Glu-Leu-Lys-Leu.
[0010] Both peptides EGEPKLP and TPELKL were identified from the neutral protein hydrolysate of shiitake mushrooms; in practice, they can also be prepared by peptide synthesis companies through chemical synthesis.
[0011] Both polypeptides provided by this invention have strong α-glucosidase inhibitory activity and are safe, non-toxic, and easily absorbed, making them suitable for use in hypoglycemic products (including hypoglycemic drugs).
[0012] Regarding the inhibitory activity of the peptides EGEPKLP and TPELKL of the present invention against α-glucosidase, the IC50 of EGEPKLP is... 50 The IC50 of TPELKL is 0.41 mg / ml. 50 The concentration was 0.34 mg / ml.
[0013] In summary, the polypeptide of the present invention has α-glucosidase inhibitory activity, which has not been reported before, and the activity effect is dose-dependent, thus belonging to a novel functional peptide with α-glucosidase activity.
[0014] The usage and dosage of the two polypeptides with α-glucosidase inhibitory activity of the present invention are as follows: oral administration, about 0.5g each time, 3 times a day. Attached Figure Description
[0015] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
[0016] Figure 1 The α-glucosidase inhibitory activity of different molecular weight lentinan enzymatic hydrolysate fractions; different letters indicate significant differences between different treatments;
[0017] Figure 2 To separate components with a molecular weight <1kDa using Sephadex G-10 gel chromatography;
[0018] Figure 2 In the middle: A. Elution curve; B. α-glucosidase inhibitory activity; different letters indicate significant differences between different treatments (P<0.05).
[0019] Figure 3 This is a mass spectrometry identification diagram. Detailed Implementation
[0020] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0021] Example 1
[0022] 1. Preparation of Lentinan
[0023] Weigh 5.0g of shiitake mushroom protein into a 150mL Erlenmeyer flask, add 100mL of distilled water and stir well. Adjust the pH to 7 and add the enzyme at a ratio of 3000U / g. -1 Add neutral protease and mix well. Place in a shaker at 45°C and rotate at 150 rpm. -1 The enzyme was hydrolyzed at 4000 rpm for 4 hours. After hydrolysis, the neutral protease was inactivated by boiling in a water bath for 5 minutes. The mixture was then cooled to room temperature and centrifuged at 4000 rpm for 20 minutes. The supernatant was then used for the next experiment.
[0024] 2. Ultrafiltration
[0025] The supernatant obtained in step 1 was separated using ultrafiltration membranes of 1 kDa, 3 kDa, and 5 kDa. The resulting fractions were named UF-I (>5 kDa), UF-II (3-5 kDa), UF-III (1-3 kDa), and UF-IV (<1 kDa), respectively, and their activities were measured.
[0026] At a concentration of 5 mg / mL, the α-glucosidase inhibitory activities of different molecular weight lentinan hydrolysate fractions were as follows: Figure 1As shown, it can be concluded that UF-Ⅳ (<1 kDa) has better activity.
[0027] 3. Gel separation
[0028] UF-Ⅳ (<1 kDa) was loaded onto a Sephadex G-10 gel filter column (1.6 × 100 cm), and then eluted with ultrapure water at a flow rate of 0.5 mL / min. One sample was collected every 10 min, and the sample was detected at 280 nm. The elution peaks were collected. Figure 2 A) and freeze-dry.
[0029] Eleven elution peaks (F1-F11) were collected. The α-glucosidase inhibitory activities of the elution peaks (F1-F11) were compared with those of... Figure 2 As shown in B, it can be concluded that component F5 has the best activity.
[0030] 4. The F5 fraction with the best activity was identified by mass spectrometry and screened using molecular docking, resulting in two active peptide sequences—EGEPKLP and TPElkL. Figure 3 .
[0031] Then, EGEPKLP and TPELKL were synthesized by chemical synthesis, and their activities were further verified.
[0032] Experiment 1: Detection method for α-glucosidase inhibitory activity involved in this invention:
[0033] Take 50 μL of a polypeptide solution of a certain concentration (prepared with 0.1 mol / L, pH 6.9 phosphate buffer) and 100 μL of 10 mg / mL α-glucosidase solution (prepared with 0.1 mol / L, pH 6.9 phosphate buffer), add them to the microplate, mix well, and incubate at 25 °C for 10 min. Then add 50 μL of 5 mmol / L PNPG (p-nitrophenyl-α-D-glucopyranoside) solution (prepared with 0.1 mol / L, pH 6.9 phosphate buffer), incubate at 37 °C for 30 min, and then add 50 μL of 0.67 mol / L Na₂CO₃ solution to terminate the reaction. Measure the absorbance at 405 nm. This system is called the sample.
[0034] The following three systems were set up: control, sample blank, and control blank.
[0035] Control: Replace 50 μL of peptide solution with 50 μL of 0.1 mol / L, pH 6.9 phosphate buffer, otherwise the same as the sample.
[0036] Sample blank: Replace 100 μL of 10 mg / mL α-glucosidase solution with 100 μL of 0.1 mol / L, pH 6.9 phosphate buffer; the rest is the same as the sample.
[0037] Control blank: The peptide solution and α-glucosidase solution were replaced with the corresponding volumes of 0.1 mol / L, pH 6.9 phosphate buffer, respectively, and the rest were the same as the sample.
[0038] The inhibition rate is calculated using the following formula.
[0039]
[0040] Example 1-1: Inhibition rate of α-glucosidase by peptide EGEPKLP at a concentration of 0.5 mg / mL
[0041] Detection method: The activity of the peptide EEGPKLP, obtained by chemical synthesis, was detected. At this time, the concentration of peptide EEGPKLP was 0.5 mg / mL.
[0042] A 对照 The average absorbance from the two measurements was 0.851, A 对照空白 The average absorbance of the two measurements was 0.033; A 样品 The absorbance values measured twice were 0.392 and 0.387, respectively. 样品空白 The average absorbance of the two measurements was 0.036;
[0043] After substituting into the above formula for calculating the inhibition rate, the inhibition rates obtained from the two measurements were 56.5% and 57.1%, with an average of 56.8%.
[0044] Results: The α-glucosidase inhibition rate of the peptide EGEPKLP at 0.5 mg / mL was 56.8%.
[0045] Examples 1-2: Inhibition rate of α-glucosidase by peptide EGEPKLP at a concentration of 0.25 mg / mL:
[0046] Detection method: The activity of the peptide EEGPKLP, obtained by chemical synthesis, was detected. At this time, the concentration of peptide EEGPKLP was 0.25 mg / mL.
[0047] A 对照 The average absorbance from the two measurements was 0.851, A 对照空白 The average absorbance of the two measurements was 0.033; A 样品 The absorbance values measured twice were 0.613 and 0.617, A 样品空白 The average absorbance of the two measurements was 0.035;
[0048] After substituting into the above formula for calculating the inhibition rate, the inhibition rates obtained from the two measurements were 29.3% and 28.9%, with an average of 29.1%.
[0049] Results: The α-glucosidase inhibition rate of the peptide EGEPKLP at 0.25 mg / mL was 29.1%.
[0050] Example 2-1: Inhibition rate of α-glucosidase by peptide TPELKL at a concentration of 0.5 mg / mL
[0051] Detection method: The activity of the peptide TPELDKL, obtained by chemical synthesis, was detected. At this time, the concentration of peptide TPELDKL was 0.5 mg / mL.
[0052] A 对照 The average absorbance from the two measurements was 0.851, A 对照空白 The average absorbance from the two measurements was 0.033, A 样品 The absorbance values measured twice were 0.311 and 0.316, A 样品空白 The average absorbance of the two measurements was 0.037;
[0053] After substituting into the above formula for calculating the inhibition rate, the inhibition rates obtained from the two measurements were 66.5% and 65.9%, with an average of 66.2%.
[0054] Results: The α-glucosidase inhibition rate of the peptide TPELKL at 0.5 mg / mL was 66.2%.
[0055] Example 2-2: Inhibition rate of α-glucosidase by peptide TPELKL at a concentration of 0.25 mg / mL
[0056] Detection method: The activity of the peptide TPELDKL, obtained by chemical synthesis, was detected. At this time, the concentration of peptide TPELDKL was 0.25 mg / mL.
[0057] A 对照 The average absorbance from the two measurements was 0.851, A 对照空白 The average absorbance from the two measurements was 0.033, A 样品 The absorbance values measured twice were 0.565 and 0.557, A 样品空白 The average absorbance of the two measurements was 0.035;
[0058] After substituting into the above formula for calculating the inhibition rate, the inhibition rates obtained from the two measurements were 35.2% and 36.2%, with an average of 35.7%.
[0059] Results: The α-glucosidase inhibition rate of the peptide TPELKL at 0.25 mg / mL was 35.7%.
[0060] Comparative example: The polypeptides obtained from the enzymatic hydrolysate of shiitake mushrooms in Table 1 were prepared into a polypeptide solution with a concentration of 0.5 mg / mL; then, the α-glucosidase inhibition rate was determined according to the above experimental method; the comparison between the results obtained and the results obtained from the polypeptides of the present invention is shown in Table 1.
[0061] Table 1
[0062] Polypeptide Alpha-glucosidase inhibition rate (%) EGEPKLP (Invention) 56.8 TPELKL (Invention) 66.2 IDEAVPR 13.2 PDDPAVVE 15.8 EEPLPQ 16.8 DPEKFP 11.5
[0063] Note: The above-mentioned IDEAVPR, PDDPAVVE, EEPLPQ, and DPEKFP are the remaining lentinan peptides obtained during the invention process.
[0064] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible, such as different structures obtained from the degradation and separation of proteins from different sources and their derived structures. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.
Claims
1. A polypeptide with α-glucosidase inhibitory activity, characterized in that it is any one of the following: The polypeptide EGEPKLP has the following amino acid sequence: Glu-Gly-Glu-Pro-Lys-Leu-Pro; The polypeptide TPELKL has the following amino acid sequence: Thr-Pro-Glu-Leu-Lys-Leu.
2. The use of the polypeptide with α-glucosidase inhibitory activity as described in claim 1 in the preparation of hypoglycemic drugs.