Alpha-glucosidase inhibitory peptide and application thereof

Abstract

The invention discloses an alpha-glucosidase inhibitory peptide and application thereof, and belongs to the technical field of biological medicine and functional food. And the sequence of the alpha-glucosidase inhibitory peptide is Ser-Pro-Tyr-Leu-Gly (SPYLG) or Tyr-Leu-Pro-Lys-Gly (YLPKG). The invention further discloses a preparation method of the alpha-glucosidase inhibitory peptide. The alpha-glucosidase inhibitory peptide has excellent biological safety. Experiments prove that the polypeptide has a remarkable inhibition capability on alpha-glucosidase and shows good gastrointestinal digestion stability. In-vivo and in-vitro studies show that the polypeptide can significantly reduce fasting blood-glucose and improve insulin resistance, the fasting blood-glucose level of diabetic mice after administration is reduced by about 50%, and liver and kidney injury caused by diabetes is effectively relieved. The polypeptide disclosed by the invention is suitable for being used as a core component for developing pharmaceutical preparations for preventing or treating type 2 diabetes and functional foods for assisting in reducing blood glucose.

Description

TECHNICAL FIELD

[0001] The application discloses an alpha-glucosidase inhibitory peptide and application thereof, and belongs to the technical field of biological medicine and functional food. BACKGROUND

[0002] Type 2 diabetes (T2DM) is a global metabolic disease characterized by high blood sugar, mainly caused by insufficient insulin secretion or insulin resistance. In the blood sugar regulation mechanism, the alpha-glucosidase of the small intestinal brush border is a key hydrolytic enzyme that breaks down complex carbohydrates into glucose into the blood. Therefore, competitively inhibiting the activity of alpha-glucosidase, delaying carbohydrate digestion and glucose absorption, is an effective strategy to reduce postprandial blood sugar.

[0003] At present, although the commonly used alpha-glucosidase inhibitors (such as acarbose) in clinical practice have definite curative effect, they are often accompanied by abdominal distension, diarrhea and other gastrointestinal adverse reactions, and long-term use may have the risk of liver damage, and the use of specific groups of people is limited. As a safe and less side-effect natural substitute, bioactive peptides have attracted much attention in recent years. However, the traditional method of obtaining active peptides from natural proteins relies on enzymatic hydrolysis, ultrafiltration and chromatographic separation, which has the technical bottlenecks of long screening period, low efficiency, high cost and lack of targeting.

[0004] Compared with traditional chemical drugs, alpha-glucosidase inhibitory peptides derived from natural proteins have significant advantages. Such active peptides not only have good biocompatibility and high safety, greatly reducing the toxic and side effects, but also can effectively avoid or alleviate the abdominal distension, diarrhea and other gastrointestinal discomfort symptoms commonly seen in acarbose and other drugs. In addition, short peptide molecules have simple molecular structures and are more easily absorbed and utilized by the human digestive tract. Therefore, as a safe, efficient and well-tolerated potential therapeutic means, alpha-glucosidase inhibitory peptides show great application prospects in the fields of diabetes prevention, functional food and development of new hypoglycemic drugs. SUMMARY

[0005] The purpose of the present application is to provide an alpha-glucosidase inhibitory peptide, which has the advantages of strong alpha-glucosidase inhibitory activity, good biocompatibility, short peptide chain sequence, simple synthesis, low cost and the like, and provides support for the development of diabetes treatment and functional food.

[0006] To achieve the purpose of the application, the following technical solutions are adopted: The 5-peptide short chain has alpha-glucosidase inhibitory activity, and the amino acid sequence is Ser-Pro-Tyr-Leu-Gly (SPYLG) or Tyr-Leu-Pro-Lys-Gly (YLPKG).

[0007] Linear peptide chain, circular peptide chain, pharmaceutically acceptable salt or derivative formed by chemical modification comprising the 5-peptide short chain.

[0008] The 5-peptide short chain is applied to the preparation of a blood sugar reducing drug, an insulin resistance improving drug and a liver and kidney damage protecting drug for diabetes.

[0009] The 5-linear peptide chain, the circular peptide chain, the pharmaceutically acceptable salt or the derivative formed by chemical modification are applied to the preparation of a blood sugar reducing drug, an insulin resistance improving drug and a liver and kidney damage protecting drug for diabetes.

[0010] The 5-peptide short chain is applied to the preparation of a health food, a special medical purpose formula food or a dietary supplement.

[0011] The beneficial effects of the present application include: 1. The peptide segments SPYLG and YLPKG provided by the present application have the advantages of safety, non-toxicity and no side effects, can effectively inhibit the activity of alpha-glucosidase, regulate postprandial blood glucose, and thus play a certain relieving effect on diabetes and its complications.

[0012] 2. The alpha-glucosidase inhibiting peptide involved in the present application can be used as a core active ingredient, and is suitable for a type 2 diabetes patient group. Compared with traditional hypoglycemic drugs, the alpha-glucosidase inhibiting peptide has significant safety and no adverse reactions.

[0013] 3. Based on a diabetes mouse model induced by high-fat feed and streptozotocin, the present application proves that the newly designed alpha-glucosidase inhibiting peptide oral solution has significant effects of reducing blood sugar and improving insulin resistance, etc. by being intervened in the form of an oral solution. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a MALDI-TOF-MS graph of the alpha-glucosidase inhibiting peptide of the present application.

[0015] Figure 2 It is an infrared spectrum graph of the alpha-glucosidase inhibiting peptide of the present application.

[0016] Figure 3 It is cytotoxicity data of the alpha-glucosidase inhibiting peptide of the present application, MTT data.

[0017] Figure 4 It is cytotoxicity data of the alpha-glucosidase inhibiting peptide of the present application, Calcein-AM / PI live and dead cell graph.

[0018] Figure 5 It is hemolysis data of the alpha-glucosidase inhibiting peptide of the present application.

[0019] Figure 6 This is the experimental data of in vitro α-glucosidase inhibition of the present invention.

[0020] Figure 7 This is the in vitro gastrointestinal digestion experimental data of the α-glucosidase inhibitory peptide of this invention.

[0021] Figure 8 This invention relates to an experiment demonstrating the inhibition of α-glucosidase by the α-glucosidase inhibitory peptide in Caco-2 cells.

[0022] Figure 9 This diagram shows the results of glucose content determination in Caco-2 cells using the α-glucosidase inhibitory peptide of this invention. In the diagram, A is the glucose standard curve; B is a histogram comparing intracellular glucose concentrations in different experimental groups.

[0023] Figure 10 This invention provides experimental data for the α-glucosidase inhibitory peptide type 2 diabetic mouse model, including fasting blood glucose and body weight measurements.

[0024] Figure 11 This is experimental data from the α-glucosidase inhibitory peptide type 2 diabetic mouse model of the present invention, including a tolerance test.

[0025] Figure 12 This is experimental data on the type 2 diabetic mouse model of α-glucosidase inhibitory peptide of the present invention, including liver and kidney function data. Detailed Implementation

[0026] The present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Preparation of α-glucosidase inhibitory peptide α-glucosidase inhibitory peptides were synthesized using the Fmoc solid-phase synthesis method. The specific steps are as follows: Dichlorotriphenylmethyl chloride resin was swollen in DCM for 0.5–3 h. The first amino acid of each of the two α-glucosidase inhibitory peptides, glycine (Gly, G), was added, and the reaction was carried out in DMF solution containing DIEA for 2 h. After the reaction, unreacted groups were blocked with methanol, and the Fmoc protecting groups were removed with 20% piperidine solution. Subsequently, following the amino acid sequence of the two α-glucosidase inhibitory peptides, SPYLG added leucine (Leu, L), tyrosine (Tyr, Y), proline (Pro, P), and serine (Ser, S) sequentially, while YLPKG added lysine (Lys, K), proline (Pro, P), leucine (Leu, L), and tyrosine (Tyr, Y) sequentially. PyBoP, HoBt, and DIEA were added to the DMF simultaneously with each amino acid addition, and the reaction was carried out for 2 h each time. After amino acid coupling, the peptides were cleaved using a cleavage reagent (trifluoroacetic acid:water:triisopropylsilane = 95:1.25:1.25, v / v / v). The peptides were precipitated in ice-cold anhydrous diethyl ether, dissolved in water, and then lyophilized. High-performance liquid chromatography (HPLC) was used to purify and analyze the crude peptides. The mobile phase ratio was adjusted to acetonitrile:water = 20:80, the flow rate was 0.2–2.0 mL / min, and the detection wavelength was 210 nm. The fractions from each peak were collected and lyophilized again.

[0028] The results are as follows Figure 1 As shown, the synthesized peptide components are uniform. The molecular weight ion peaks of SPYLG (A) and YLPKG (B) in MALDI-TOF-MS detection are 536 Da (C) and 577 Da (D), respectively. They have high purity and only have a single chromatographic peak, which can be used for subsequent experiments. Example 2: Structural identification of α-glucosidase inhibitory peptide Infrared spectroscopy analysis: An appropriate amount of lyophilized peptide sample was mixed with spectral-grade potassium bromide in an agate mortar and ground into an extremely fine powder, then pressed into a translucent sheet. The prepared potassium bromide sheet was placed into the sample cell of the infrared spectrometer. The testing environment was maintained at room temperature and in a dry air atmosphere. The instrument scanning wavenumber range was set to 4000–400 cm⁻¹, the spectral resolution was set to 4 cm⁻¹, and the cumulative number of scans was 32. During the test, a pure potassium bromide blank sheet was used as a reference to correct for background interference, thereby obtaining the infrared absorption spectrum of the peptide sample and analyzing the peak positions of its characteristic functional groups.

[0029] The results are as follows Figure 2 As shown, the two peptide chains exhibit characteristic broad peaks in different regions. The presence of these different characteristic peaks confirms the formation of peptide bond structures in the product, which is consistent with the chemical structural characteristics of the expected target α-glucosidase inhibitory peptide. Example 3: Cytotoxicity of α-glucosidase inhibitory peptides Three cell lines, L-929, HepG2, and Caco-2, were selected as models. Cells were collected when they reached 70%-80% confluence. (1) Quantitative analysis: The collected cells were plated in 96-well plates and adhered for 24 h. Then, they were treated with different concentrations of peptides for 24 h. MTT reagent was then added and reacted for 4 h in the dark. DMSO was then added, and the absorbance at 490 nm was measured to quantify cell viability. (2) Qualitative observation: Cells were seeded in confocal culture dishes and subjected to 24 h of adhesion and peptide treatment in the same manner. Calcein-AM / PI staining solution was then added and stained for 20 min in the dark. The microscopic morphology and survival distribution of the cells after drug administration were directly observed using a confocal microscope.

[0030] The results are as follows Figure 3 As shown, the survival rate of the three cell types (L-929 / HepG2 / Caco-2) was above 87% under the intervention of two α-glucosidase inhibitory peptides, and... Figure 4 The results of live and dead cell staining showed that the cells in the drug-treated group had good morphology, which indicates that SPYLG and YLPKG have good biocompatibility. Example 4: Hemolytic activity of α-glucosidase inhibitory peptides Fresh blood was repeatedly washed with PBS buffer to remove plasma and leukocytes until the supernatant was clear. Then, erythrocytes were mixed with equal volumes of peptide solutions of different gradients and incubated at 37 °C. After centrifugation at 3000 rpm for 3 min, the supernatant was collected and its absorbance was measured at 540 nm. Triton X-100 was used as a complete hemolysis control to assess the degree of hemolysis induced by the peptides. The centrifuged cell pellet was fixed with 2.5% glutaraldehyde and dehydrated by a gradient ethanol exchange. Finally, the effects of the peptides on the blood cell membrane structure were visually analyzed using scanning electron microscopy (SEM).

[0031] The results are as follows Figure 5 As shown, the hemolysis rates of SPYLG and YLPKG were both below 5%, and the blood cell structure remained intact under SEM, indicating that the α-glucosidase inhibitory peptides are safe for hemolysis. Example 5: In vitro α-glucosidase inhibition experiment A series of concentration solutions (10, 25, 50, 100, 1000 μg / mL) of α-glucosidase inhibitory peptide were prepared by dissolving the peptide in PBS buffer. These solutions were then incubated with α-glucosidase solution (0.2 U / mL) at a volume ratio of 1:1 at 37 °C for 15 min. Acarbose was used as the positive control. The substrate pNPG was then added, and the reaction was continued at 37 °C for another 15 min. Finally, 0.10 mol / L Na₂CO₃ was added to terminate the reaction, and the absorbance was measured at 405 nm. A buffer solution was used instead of the sample as a blank control. The α-glucosidase inhibition rate was calculated using the formula: α-glucosidase inhibition rate (%) = [(A blank - A sample) / A blank] x 100.

[0032] The results are as follows Figure 6 As shown, the α-glucosidase inhibitory peptides SPYLG and YLPKG of the present invention both exhibit significant inhibitory activity (as shown in A). Their IC50 values... 50 The values ​​were all lower than those of the positive control acarbose (such as B, C, and D), indicating that the two peptides have excellent hypoglycemic effects and great potential for drug development. Example 6: In vitro gastrointestinal digestion experiment An in vitro tandem digestion system incorporating both gastric and intestinal phases was constructed. Synthetic peptides were first incubated in a simulated gastric environment at 37°C for 240 min with periodic sampling. The pH was then adjusted to 7.0-8.0, and an equal volume of simulated intestinal fluid was introduced to continue the reaction. After the entire process, residual enzymes were inactivated at 95°C for 10 min. Multidimensional coupled-array techniques were used to analyze the digestion products. HPLC was used to determine the remaining percentage of intact peptides, combined with UV-Vis spectroscopy to monitor the balance of total substances and the evolution of spectroscopic characteristics. Furthermore, the structure-activity relationship stability of the peptides during gastrointestinal transport was verified by comparing the α-glucosidase inhibitory activity of samples before and after digestion.

[0033] Figure 7The stability of the α-glucosidase inhibitory peptides in in vitro simulated gastrointestinal digestion is shown. A represents the change in α-glucosidase inhibitory activity of SPYLG with digestion time (0-4 h); B is the UV-Vis absorption spectrum of SPYLG; C is the HPLC chromatogram of SPYLG at different digestion time points; D is the peak area of ​​SPYLG based on HPLC; E represents the change in α-glucosidase inhibitory activity of YLPKG with digestion time (0-4 h); F is the UV-Vis absorption spectrum of YLPKG; G is the HPLC chromatogram of YLPKG at different digestion time points; and H is the peak area of ​​YLPKG based on HPLC. The results demonstrate that the α-glucosidase inhibitory peptides SPYLG and YLPKG of this invention exhibit good anti-enzymatic stability in in vitro simulated gastrointestinal fluid. They maintain high structural integrity and retain significant biological activity in the digestive tract environment. Example 7: Inhibition of α-glucosidase in Caco-2 cells Caco-2 cells cultured to full differentiation in 24-well plates were used as an intestinal enzyme model. Cells were randomly grouped and pretreated with a series of concentrations of synthetic α-glucosidase inhibitory peptides. Polysaccharides were then added as substrates, and the mixture was co-incubated at 37 °C for in situ enzymatic digestion. After the reaction, the supernatant was rapidly separated and heat-inactivated to prepare the sample for testing.

[0034] The results are as follows Figure 8 As shown, the α-glucosidase inhibitory peptide of the present invention also exhibits significant α-glucosidase inhibitory activity in the Caco-2 cell environment (e.g., A). More importantly, the cell-level IC50 values ​​of SPYLG and YLPKG... 50 The values ​​were all significantly lower than those of acarbose (e.g., B, C, D). This indicates that the α-glucosidase inhibitory peptide of this invention achieves superior hypoglycemic potential at the cellular level compared to traditional drugs. Example 8: Detection of intracellular glucose in Caco-2 cells The glucose content in samples from Caco-2 cells was analyzed. Following the instructions of the glucose assay kit, the inactivated supernatant was mixed with the assay reagent for color development. The optical density was measured spectrophotometrically, and the endpoint glucose concentration in each well was calculated. This method was used to quantify the catalytic efficiency of α-glucosidase in converting polysaccharides to glucose, thereby evaluating the hypoglycemic activity of the target peptide.

[0035] Figure 9This diagram shows the results of glucose content determination in Caco-2 cells using the α-glucosidase inhibitory peptides of this invention. A is the glucose standard curve; B is a histogram comparing intracellular glucose concentrations in each experimental group. The α-glucosidase inhibitory peptides SPYLG and YLPKG of this invention can effectively block the abnormal increase in intracellular glucose in Caco-2 cells. They can reduce substrate hydrolysis and decrease glucose production and absorption by inhibiting the activity of α-glucosidase on the surface of intestinal cells.

[0036] Example 9: Type 2 Diabetic Mouse Model Age-appropriate C57BL / 6 mice were induced with a 60% high-fat diet (HFD) for 4-8 weeks, followed by intraperitoneal injection of streptozotocin (STZ) for 3 consecutive days to establish a type 2 diabetes mellitus (T2DM) model. Mice were then randomly divided into a model group (DM), a normal control group (NC), a peptide-treated group (SPYLG, YLPKG), and a positive control group (acarbose). The α-glucosidase inhibitory peptide was dissolved in physiological saline to prepare an oral solution, which was administered to mice by gavage for 28 consecutive days. During the treatment period, fasting blood glucose (FBG) and body weight were monitored weekly after fasting. Starch tolerance tests (OSTT), oral glucose tolerance tests (OGTT), and insulin tolerance tests (ITT) were performed during this period, and blood glucose was measured at multiple time points within 0-120 minutes to calculate the area under the curve (AUC). Serum samples were collected to measure liver and kidney function indicators (ALT, AST, Urea, Creatinine).

[0037] The results are as follows Figure 10 The results show that the α-glucosidase inhibitory peptide has a long-term and stable hypoglycemic effect. A represents blood glucose concentration and B represents body weight. To a certain extent, it alleviates metabolic disorders and emaciation caused by the disease and maintains a relatively stable physiological state. Figure 11 This demonstrates that the α-glucosidase inhibitory peptide can significantly improve glucose homeostasis in mice. A is the OSTTT, B is the AUC calculated based on the OSTTT; C is the OGTT, D is the AUC calculated based on the OGTT; E is the ITT, F is the AUC calculated based on the ITT. In mice administered SPYLG and YLPKG, peak blood glucose levels decreased, and the AUC was significantly reduced, with inhibitory effects comparable to the positive control acarbose. In the ITT test, the α-glucosidase inhibitory peptide group showed a significantly greater decrease in blood glucose than the DM group, indicating that the α-glucosidase inhibitory peptide of this invention can effectively improve insulin resistance in diabetic mice and enhance the body's sensitivity to insulin. Figure 12 The study also measured liver and kidney function indicators in different mice, where A represented ALT, B represented AST, C represented Urea, and D represented Creatinine. This indicates that the α-glucosidase inhibitory peptide not only possesses hypoglycemic activity but also exhibits significant hepatoprotective and renal protective functions. It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A short pentapeptide chain with α-glucosidase inhibitory activity, characterized in that: Its amino acid sequence is Ser-Pro-Tyr-Leu-Gly (SPYLG) or Tyr-Leu-Pro-Lys-Gly (YLPKG).

2. A linear peptide chain, a cyclic peptide chain, a pharmaceutically acceptable salt thereof, or a derivative thereof formed by chemical modification comprising the 5-peptide short chain of claim 1.

3. The use of the pentapeptide short chain according to claim 1 in the preparation of hypoglycemic drugs, drugs to improve insulin resistance, and drugs to protect against diabetic liver and kidney damage.

4. The use of the 5-linear peptide chain, cyclic peptide chain, pharmaceutically acceptable salt thereof, or derivative thereof formed by chemical modification as described in claim 2, in the preparation of hypoglycemic drugs, drugs to improve insulin resistance, and drugs to protect against diabetic liver and kidney damage.

5. The application of the 5-peptide short chain according to claim 1 in the preparation of health foods, special medical purpose formula foods or dietary supplements.

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