Hypoglycemic peptide and application thereof

Abstract

The invention discloses a hypoglycemic peptide and application thereof, and belongs to the technical field of active peptides. The hypoglycemic peptide comprises peptides of which the sequences are respectively DSVF (Asp-Ser-Val-Phe) and / or TLAE (Thr-Leu-Ala-Glu). The invention further discloses a preparation method of the hypoglycemic peptide. Animal experiments prove that the hypoglycemic peptide can assist in reducing blood sugar and improve cognitive impairment of diabetes mellitus, so that the hypoglycemic peptide can be used for preparing drugs for reducing blood sugar and improving cognitive impairment of diabetes mellitus.

Description

Technical Field

[0001] The invention relates to a glucose-lowering peptide and application thereof, belonging to the technical field of active peptides. Background Art

[0002] Diabetes is a metabolic disease characterized by insufficient secretion or utilization of insulin and high blood sugar. Epidemiological data show that as of 2021, the number of people with diabetes worldwide is about 537 million, and it is expected to rise to 643 million by 2030. Long-term hyperglycemia in diabetic patients can lead to the development of a variety of complications such as diabetic nephropathy, diabetic foot, retinopathy and cognitive dysfunction. Cognitive dysfunction refers to the impairment of one or more of memory, language, visual space, execution, calculation and understanding judgment. Its clinical manifestations include memory loss, decreased learning ability, language, judgment and perceptual motor impairment. It is a serious complication with a high incidence rate in diabetic patients. The longer the course of diabetes, the worse the cognitive function. Cognitive dysfunction will aggravate the patient's reduced self-management ability, forming a vicious circle and bringing heavy burdens to society, families and patients. The current treatment methods for diabetic cognitive dysfunction mainly use drugs such as blood sugar control drugs, cerebral circulation improvers, and brain metabolism improvers. Clinical treatment focuses on controlling blood sugar levels and reducing insulin resistance. Among them, the main drugs for controlling blood sugar include acarbose, voglitazone and metformin, but they will inevitably cause adverse reactions such as hypoglycemia, weight gain, diarrhea or nausea, and in severe cases, they will also cause liver and kidney damage in patients. Long-term medication for elderly people with underlying diseases has a high safety risk. Therefore, it is necessary to develop safe and effective health foods that assist in lowering blood sugar and improving the memory function of diabetic patients, as well as drugs that intervene in cognitive dysfunction in diabetic patients.

[0003] Patent publication number CN118271398A discloses a porcine collagen-derived glucose-lowering peptide, which uses pig skin as raw material, extracts collagen through ultrasound-assisted acid method, and then obtains glucose-lowering peptide through pepsin hydrolysis. It can promote the secretion of insulin by Min6 cells, improve the absorption of glucose by pancreatic β cells and exert a glucose-lowering effect. Patent publication number CN111607628A uses protease hydrolysis, membrane separation, activated carbon decolorization, deodorization and spray drying technology to prepare sea yam and camel blood glucose-lowering mixed peptides, respectively, and mixes the two in a ratio of 1-1.5:1 to prepare a sea yam and camel blood glucose-lowering mixed peptide. Sea yam and camel blood glucose-lowering mixed peptides inhibit α-amylase IC 50 =0.5mg / mL, IC for α-glucosidase inhibition 50 The IC value of DPP-IV inhibition was 51 μg / mL. 50It is 43μg / mL, which has good hypoglycemic activity in vitro. At present, the main method is to extract the protein and then add protease to hydrolyze the raw material, and then obtain the hypoglycemic activity mixed peptides through a series of separation and purification. The operation process is relatively complicated, the peptide sequence is unclear, and the in vitro activity evaluation is mainly used. Whether it has hypoglycemic activity in vivo is unclear. The patent with publication number CN117778511A discloses a preparation method and application of royal jelly protein hypoglycemic peptide powder. The protein in fresh royal jelly is used as the raw material, and the royal jelly mixed peptide powder is obtained by three enzymatic hydrolysis of alkaline protease, pepsin and trypsin, and freeze-drying. The key efficacy peptides are identified by calculating the enzymatic peptide segment, combining the glycosidase peptide library and the LBSPepPredictor tool to obtain GSR, LW, IW, TW, IF, PR and CL7 highly active peptide segments, among which PR shows strong α-glucosidase inhibitory activity (19.79μmol / L). The preparation steps of this invention are complicated, and it is necessary to obtain a hypoglycemic activity mixed peptide by three enzymatic hydrolysis and then freeze-drying, and the in vitro hypoglycemic activity evaluation is mainly used. Whether it has hypoglycemic activity in vivo is still unclear. The glucose-lowering peptides reported so far mainly focus on assisting blood sugar lowering through α-glucosidase inhibitory activity, and there is a lack of glucose-lowering peptides that can regulate blood sugar and intervene in and improve diabetic cognitive dysfunction. Summary of the invention

[0004] One of the objects of the present invention is to provide a glucose-lowering peptide, which comprises a peptide having an amino acid sequence of Asp-Ser-Val-Phe (DSVF) and / or Thr-Leu-Ala-Glu (TLAE).

[0005] The glucose-lowering peptide of the present invention is extracted from ham.

[0006] Preferably, the ham is Xuanwei ham and Wenshan ham.

[0007] Preferably, a method for preparing a glucose-lowering peptide comprises the following specific steps:

[0008] (1) Weigh the ham, mince it, add phosphate buffer, centrifuge and collect the supernatant, add ethanol solution to the supernatant, let it stand at 4°C for 12 hours, centrifuge and collect the supernatant, ultrafilter the supernatant with a filter membrane with a molecular weight cutoff of 10KDa, collect the filtrate, and obtain the crude ham hypoglycemic peptide.

[0009] (2) Using liquid chromatography-mass spectrometry (LC-MS / MS) to purify the crude ham hypoglycemic peptide obtained in step (1) and identify the peptide amino acid sequence, two peptides with amino acid sequences of Asp-Ser-Val-Phe (DSVF) and Thr-Leu-Ala-Glu (TLAE) were obtained.

[0010] Preferably, in step (1), the mass volume ratio of ham to phosphate buffer is: (1-3): (2-20), expressed in g:ml, and the pH of the phosphate buffer is 7.4.

[0011] Preferably, the centrifugation condition in step (1) is 12000 rpm / min at 4°C for 20 min.

[0012] Preferably, in step (1), the volume ratio of the ethanol solution to the phosphate buffer is (3-30):(1-10), and the mass concentration of the ethanol solution is 40%.

[0013] Preferably, step (2) is purified and analyzed using Q-Exactive LC-MS / MS, and the amino acid sequence of the glucose-lowering peptide is identified using PeaksStudio software. Agilent Eclipse Plus C18 column (2.1 mm × 150 mm, 3.5 μm) was used for separation and purification. The mobile phase A was water: formic acid = 1000: 1 (V / V), and the mobile phase B was acetonitrile: formic acid = 1000: 1 (V / V). 0-5 min was eluted with 5% of the phase B, 5-10 min was eluted with 5%-20% of the phase B, 10-25 min was eluted with 20%-40% of the phase B, 25-35 min was eluted with 40%-75% of the phase B, and 35-40 min was eluted with 5% of the phase B. The flow rate was 0.2 mL / min, the column temperature was 30 °C, the injection volume was 5 μL, the injection concentration was 0.1 mg / mL, the detection wavelength was 220 nm, and the mass spectrometry resolution was Full MS 35000, mass spectrometry detection was performed in positive ion mode with a mass range of 100-2000 m / z and collision energies of 10 eV, 20 eV, and 30 eV.

[0014] The third object of the present invention is to provide a use of the glucose-lowering peptide in the preparation of a drug for improving diabetes and cognitive dysfunction in diabetic patients.

[0015] Beneficial effects of the present invention

[0016] (1) Animal experiments have shown that the novel glucose-lowering peptide with an amino acid sequence described in the present invention can lower blood sugar and improve cognitive dysfunction in diabetic patients.

[0017] (2) The glucose-lowering peptide of the present invention can be extracted from ham. The raw material source is simple and safe and can be prepared by conventional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the trajectory diagram of the open field test of mice in the DSVF intervention group ( Figure 1 (A)) and central area exploration time histogram ( Figure 1 (B)), histogram of erection and modification times ( Figure 1 (C)).

[0019] Figure 2 This is the trajectory diagram of the open field test of mice in the TLAE intervention group ( Figure 2 (A)) and central area exploration time histogram ( Figure 2 (B)), histogram of erection and modification times ( Figure 2 (C)).

[0020] Figure 3 This is the trajectory diagram of the new foreign body recognition experiment of mice in the DSVF intervention group ( Figure 3 (A)) and NOI identification index histogram ( Figure 3 (B)).

[0021] Figure 4 This is the experimental trajectory diagram of new foreign body recognition in mice in the TLAE intervention group ( Figure 4 (A)) and NOI identification index histogram ( Figure 4 (B)).

[0022] Figure 5 The effect of DSVF on the release levels of CRH, ACTH and CORT in the HPA axis of mice.

[0023] Figure 6 The effect of TLAE on the release levels of CRH, ACTH and CORT in the HPA axis of mice. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0025] Example 1

[0026] Preparation and screening of glucose-lowering peptides.

[0027] (1) Weigh 10 g of Xuanwei ham, mince it with a blender, add 40 ml of phosphate buffer (pH 7.4), centrifuge at 12,000 rpm / min at 4°C for 20 min, collect the supernatant, add 120 ml of 40% ethanol solution to the supernatant, let it stand at 4°C for 12 h, centrifuge at 12,000 rpm / min at 4°C for 20 min, and collect the supernatant.

[0028] (2) Weigh 20 g of Wenshan ham, mince it with a blender, add 100 ml of phosphate buffer (pH 7.4), centrifuge at 12,000 rpm / min at 4°C for 20 min, and collect the supernatant. Add 300 ml of 40% ethanol solution, let stand at 4°C for 12 h, centrifuge at 12,000 rpm / min at 4°C for 20 min, and collect the supernatant.

[0029] (3) The supernatants collected in step (1) and step (2) are respectively ultrafiltered using a filter membrane with a molecular weight cutoff of 10 KDa, and the filtrates are collected to obtain Xuanwei ham hypoglycemic crude peptides and Wenshan ham hypoglycemic crude peptides.

[0030] (4) Purification and analysis were performed using Q-Exactive LC-MS / MS, and the amino acid sequence of the glucose-lowering peptide was identified using PeaksStudio software. The peptide was separated and purified using an Agilent Eclipse Plus C18 column (2.1 mm × 150 mm, 3.5 μm), with mobile phase A consisting of water:formic acid = 1000:1 (V / V), mobile phase B consisting of acetonitrile:formic acid = 1000:1 (V / V), elution with 5% B phase from 0 to 5 min, 5%-20% B phase from 5 to 10 min, 20%-40% B phase from 10 to 25 min, 40%-75% B phase from 25 to 35 min, and 5% B phase from 35 to 40 min, with a flow rate of 0.2 mL / min, a column temperature of 30°C, an injection volume of 5 μL, an injection concentration of 0.1 mg / mL, and a detection wavelength of 220 nm. The mass spectrometry resolution was Full MS 35000, mass spectrometry detection was performed in positive ion mode, the mass range was 100-2000 m / z, and the collision energy was 10 eV, 20 eV, and 30 eV. The polypeptide sequences identified from the crude glucose-lowering peptides of Xuanwei ham are Asp-Ser-Val-Phe (DSVF), Ala-Phe-Val-Thr (AFVT), Leu-Leu-Leu-Pro-Lys-Pro-Leu (LLLPKPL) and Leu-Val-Ala-Leu (LVAL); the polypeptide sequences identified from the crude glucose-lowering peptides of Wenshan ham are Thr-Leu-Ala-Glu (TLAE), Val-Val-Pro-Val-Pro (VVPVP), Leu-Leu-Phe-Val-Leu-Pro-Leu (LLFVLPL), Ala-Pro-Pro-Pro-Pro (APPP) and Leu-Leu-Leu-Cys-Ala-Phe (LLLCAF).

[0031] (5) Molecular docking SYBYL-X 2.1.1 software was used to draw the three-dimensional structure of the glucose-lowering peptide. The three-dimensional structure files of α-amylase (PDB ID: 1PPI) and DPP-IV (PDB ID: 4a5s) were searched and downloaded from the PDB protein database. The receptor protein was hydrogenated and all water molecules were removed. The glucose-lowering peptide obtained in step (4) was subjected to molecular docking analysis with the two proteins (α-amylase and DPP-IV). The sites and number of hydrogen bonds of the receptor-ligand interaction were calculated. The four peptides Asp-Ser-Val-Phe (DSVF), Thr-Leu-Ala-Glu (TLAE), Ala-Phe-Val-Thr (AFVT) and Leu-Leu-Leu-Pro-Lys-Pro-Leu (LLLPKPL) with the top four sites and hydrogen bonds were selected (Table 1).

[0032] (6) Peptide synthesis: The four peptides selected in step (5) were commissioned to Nanjing Jiepeptide Biotechnology Co., Ltd. for solid phase synthesis and desalting, with a purity of ≥98%.

[0033] (7) The in vitro hypoglycemic activity of the four ham peptides was evaluated by measuring the inhibition rate of α-amylase and DPP-IV.

[0034] α-Amylase inhibition rate determination: The four peptides synthesized on the solid phase in step (6) were prepared into solutions with a concentration gradient of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL, and acarbose at the same concentration was used as a positive control.

[0035] Add 0.4 mL of sample and 0.4 mL of α-amylase (700 U / g) to a 2 mL centrifuge tube, mix well, incubate in a 37 °C water bath for 10 min, add 0.4 mL of soluble starch (1%), continue incubation for 3 min, add 0.4 mL of DNS reagent, boil for 10 min, cool rapidly, and measure the absorbance at 540 nm, which is recorded as A. 样品 .

[0036] Phosphate buffer was used instead of DNS and the absorbance value was recorded as A 对照 ;

[0037] Ultrapure water was used to replace the sample and the absorbance value was recorded as A. 空白 ;

[0038] Acarbose was used instead of the sample as a positive control.

[0039] The calculation formula of α-amylase inhibition rate is:

[0040]

[0041] The above five concentrations of peptide solutions were compared with the corresponding α-amylase inhibition rates to draw a standard curve to obtain the 50% inhibition rate concentration of α-amylase, i.e., IC 50 value.

[0042] DPP-IV inhibition rate determination: The four peptides synthesized on the solid phase in step (6) were prepared into solutions with a concentration gradient of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL, and sitagliptin at the same concentration was used as a positive control. All reagents in the experiment were prepared with 100 mM Tris-buffer (pH 8.0).

[0043] 40 μL of DPP-Ⅳ (100 ng / mL), 200 μL of substrate Gly-Pro-pNA (500 μM) and 80 uL of sample were mixed, incubated at 37°C for 1 h, 200 μL of the mixture was pipetted into a 96-well plate, and the absorbance was measured at 405 nm.

[0044] Tris-buffer was used to replace the samples as the control group;

[0045] Tris-buffer buffer replaces enzyme as color set,

[0046] Sitagliptin substituted sample was used as positive control.

[0047]

[0048] A standard curve was drawn between the above five peptide solutions and the corresponding DPP-IV inhibition rates to obtain the DPP-IV 50% inhibition rate concentration, ie, the IC50 value.

[0049] The inhibition rates of α-amylase and DPP-IV in vitro were analyzed (Table 2), and the most active DSVF (Asp-Ser-Val-Phe) and TLAE (Thr-Leu-Ala-Glu) were obtained, which are glucose-lowering peptides.

[0050] Table 1 Docking results of glucose-lowering peptides with α-amylase and DPP-IV

[0051]

[0052] Table 2 Inhibition rate of glucose-lowering peptides on α-amylase and DPP-IV

[0053] Peptide sequence <![CDATA[α-Amylase inhibition rate IC 50 (mg / mL)]]> <![CDATA[DPP-IV inhibition rate IC 50 (mg / mL)]]> DSVF 2.25 0.56 TLAE 2.37 0.64 AFVT 2.41 0.70 LLLPKPL 4.45 1.07

[0054] Example 2

[0055] Effect of glucose-lowering peptide on improving cognitive dysfunction in T2DM mice (type 2 diabetic mice)

[0056] (1) Establishment of T2DM mouse model: Forty male C57BL / 6J mice aged 4-5 weeks (weight 28-20 g) were randomly divided into a normal group (Control), a diabetes model group (Model), a diabetes + acarbose intervention group (Aca), a diabetes + low-dose DSVF intervention group (DL), a diabetes + high-dose DSVF intervention group (DH), a diabetes + low-dose TLAE intervention group (TL), and a diabetes + high-dose TLAE intervention group (TH). During the entire experiment, the mice in the Control group were fed with ordinary feed, and the diabetic mice were fed with high-sugar and high-fat feed for 7 weeks. After that, the mice were fasted overnight but not water. Subsequently, the diabetic mice were intraperitoneally injected with glucosamine-nitrosourea (STZ) solution (40 mg / kg, dissolved in 0.1 mol / L citric acid buffer, pH 4.5) three times continuously, with an interval of 48 h between each injection. The fasting blood glucose level of the mice was measured on the second day after the injection. When the blood glucose level was greater than 7.8 mmol / L, they were considered as T2DM mice. Mice whose blood sugar levels did not meet the standard continued to be injected with STZ solution until their blood sugar reached the standard. After ensuring that the modeling of each group of mice was successful, the mice in the normal group continued to be fed with ordinary feed, and the mice in the other groups were fed with high-sugar and high-fat feed, and gavage intervention was performed. The mice in the acarbose intervention group were gavaged with 10 mg / kg acarbose (Aca) every day. The mice in the low-dose intervention group and the high-dose intervention group of glucose-lowering peptide were gavaged with 5 mg / kg and 10 mg / kg glucose-lowering peptide, respectively, every day for 4 weeks. Fasting blood sugar was monitored every week, and open field test and new foreign body recognition test were performed after 4 weeks of monitoring.

[0057] Table 3 shows that the fasting blood glucose of the diabetes model group was maintained between 12.40 and 13.40, indicating that it was always in a state of hyperglycemia. In the first week of gavage of Aca and glucose-lowering peptides to T2DM mice, the fasting blood glucose of other intervention groups decreased except for the TL group, and the fasting blood glucose of each intervention group gradually decreased during the continuous gavage process. The fasting blood glucose of the Aca group reached normal levels after 3 weeks of gavage. After 4 weeks of gavage, the fasting blood glucose of DL, DH, TL and TH mice decreased significantly compared with the Model group. Among them, the fasting blood glucose of the DH and TH groups was 7.80mmol / L and 7.40mmol / L respectively from the first week, both reaching normal levels. The results of fasting blood glucose monitoring showed that glucose-lowering peptides have the effect of regulating the fasting blood glucose of T2DM mice.

[0058] Table 3 Weekly fasting blood glucose values ​​of mice under the intervention of glucose-lowering peptides

[0059]

[0060] The open field test was used to test the free exploration ability of mice. The length, width and height of the open field were 50*50*40 cm respectively. The center area was located using the central quarter of the bottom. Each mouse was placed in the open field from the center position and allowed to explore freely for 3 minutes.

[0061] The cognitive ability of mice was assessed by the novel foreign object recognition test using the difference in exploration time between familiar objects and novel objects. Two objects (A and B) of the same size, color and shape were placed in an open field and the two objects were fixed. In the early stage of the experiment, the mice were placed in an open field to explore freely for 10 minutes, and then returned to the cage to rest. After 6 hours, object B was replaced with object C with different color and shape, and the mice were placed in the same field in the same way to explore freely for 10 minutes. After each mouse finished exploring, the field was cleaned with 75% alcohol. The activity trajectory of the mice was recorded using a camera.

[0062] (2) After 4 weeks, the mice were killed by cervical dislocation, and 0.1 g of plasma, brain, and intestinal tissue were collected and homogenized with 0.9 mL of normal saline at low temperature. The mixture was centrifuged at 9000 rpm / min for 15 min, and the supernatant was used to measure the levels of CRH (corticotropin-releasing hormone), ACTH (adrenocorticotropic hormone), and CORT (corticosterone) in serum, brain, and intestinal tissue according to the instructions of the kit.

[0063] Results Analysis

[0064] Compared with the mice in the Control group, the total travel distance and exploration time of the mice in the Model group in the central area were significantly reduced ( Figure 1 (B)), the number of erections and modifications increased significantly ( Figure 1 (C)), the exploration time of the central area in the Aca group, DL and DH intervention groups increased significantly ( Figure 1 (B)), the number of erections and modifications decreased significantly ( Figure 1 (C)). The mouse activity trajectory diagram shows that compared with the Model group, the free exploration ability of mice in the Aca group, DL and DH intervention groups increased ( Figure 1 (A)).

[0065] like Figure 2 The results showed that the activity distance and the time spent in the central area of ​​the Model group mice were significantly reduced ( Figure 2 (B)), the number of erections and modifications increased significantly ( Figure 2 (C)) was significantly different from the control group. After Aca, TL and TH intervention, the mice's residence time in the central area (Aca group: 16.27s, TL group: 16.49s, TH group: 19.09s) was significantly increased compared with the model group (9.06s). Figure 2(B)), the number of upright and modified times decreased from 29.00 in the Model group to 20.00 in the Aca group, 20.00 in the TL group, and 19.00 in the TH group, with significant differences ( Figure 2 (C)). The mouse activity trajectory diagram shows that compared with the Model group, the free exploration ability of mice in the Aca group, TL and TH intervention groups increased ( Figure 2 (A)). The results showed that DSVF and TLAE could improve the free exploration ability of T2DM mice.

[0066] The results of the novel foreign body recognition experiment of mice in the DSVF intervention group were as follows: Figure 3 As shown, compared with the Control group, the novelty identification index (NOI) of the Model group decreased by nearly half ( Figure 3 (B)), indicating that the Model group mice could not distinguish between new objects and familiar objects. The NOI of the Aca group, DL and DH intervention groups increased significantly ( Figure 3 (B)).

[0067] The results of the new foreign body recognition experiment of mice in the TLAE intervention group are as follows Figure 4 As shown, except for the Model group, the discrimination index of mice was less than zero, while the discrimination indexes of the other groups were greater than zero ( Figure 4 (B) ), the DI of mice in the Control group was the largest, which was 0.46, followed by the TL group with 0.42, the TH group with 0.33, and the Aca group with 0.21. There was no significant difference among the three groups, but the difference was significant with the Model group. The results showed that DSVF and TLAE can alleviate the cognitive dysfunction of the difference in exploration time of familiar objects and novel objects in T2DM mice.

[0068] Compared with the Control group, the levels of CRH, ACTH and CORT in the Model group were significantly increased. Acarbose and low- and high-dose DSVF intervention significantly reduced the levels of CRH, ACTH and CORT in the serum, brain tissue and intestinal tissue of mice ( Figure 5 ).

[0069] Effects of TLAE on CRH, ACTH and CORT levels in the HPA axis of T2DM mice Figure 6 As shown in the figure, compared with the Control group, the levels of CRH, ACTH and CORT in the serum, brain tissue and intestinal tissue of the Model group mice were significantly increased. After intervention with different doses of TLAE, the levels of CRH, ACTH and CORT in the serum, brain tissue and intestinal tissue of T2DM mice were significantly decreased, which was significantly different from the Model group. The results showed that DSVF and TLAE can improve the HPA axis dysfunction in T2DM mice.

[0070] In summary, the glucose-lowering peptide of this embodiment has the potential to be developed into a drug that assists in lowering blood sugar, improves the memory function of diabetic patients, and intervenes in cognitive dysfunction in diabetic patients.

Claims

1. A glucose-lowering peptide, characterized in that: The glucose-lowering peptide comprises a peptide having an amino acid sequence of Asp-Ser-Val-Phe and / or Thr-Leu-Ala-Glu.

2. The glucose-lowering peptide according to claim 1 is extracted from ham.

3. Use of the glucose-lowering peptide according to claim 1 in the preparation of a drug for improving diabetes.

4. Use of the glucose-lowering peptide according to claim 1 in the preparation of a drug for improving diabetic cognitive dysfunction.

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