Hypoglycemic peptide and application thereof

Abstract

The application discloses a hypoglycemic peptide and application thereof, and belongs to the technical field of active peptides. The hypoglycemic peptide comprises a peptide with sequences of DSVF (Asp-Ser-Val-Phe) and / or TLAE (Thr-Leu-Ala-Glu). Animal experiments prove that the hypoglycemic peptide can assist in reducing blood sugar and improving cognitive dysfunction of diabetes, and therefore the hypoglycemic peptide can be used for preparing a medicine for reducing blood sugar and improving cognitive dysfunction of diabetes.

Description

TECHNICAL FIELD

[0001] The present application relates to a hypoglycemic peptide and its application, belonging to the technical field of active peptides. BACKGROUND

[0002] Diabetes mellitus is a metabolic disease characterized by insufficient insulin secretion or utilization with high blood glucose. Epidemiological data shows that as of 2021, the global prevalence of diabetes is about 537 million, and it is expected to rise to 643 million by 2030. The long-term high blood glucose state of diabetic patients can lead to the development of various complications such as diabetic nephropathy, diabetic foot, retinopathy, and cognitive dysfunction. Cognitive dysfunction refers to the impairment of one or more aspects of memory, language, visual space, execution, calculation, and understanding and judgment, and its clinical manifestations include memory loss, decreased learning ability, impaired language, judgment, and perceptual motor skills. It is a serious complication with a high incidence in diabetic patients. The longer the duration of diabetes, the worse the cognitive function, and cognitive dysfunction will exacerbate the patient's self-management ability, forming a vicious cycle and causing a heavy burden on society, family, and patients. The current treatment methods for diabetic cognitive dysfunction mainly use blood glucose control drugs, brain circulation improvers, and brain metabolism improvers. The clinical treatment mainly controls blood glucose levels and reduces insulin resistance. Among them, the drugs for controlling blood glucose mainly include acarbose, voglibose, and metformin, etc. However, they inevitably cause adverse reactions such as hypoglycemia, weight gain, diarrhea, or nausea, and in severe cases, they can also cause liver and kidney function damage in patients, posing a high safety risk for long-term medication in elderly patients with underlying diseases. Therefore, it is necessary to develop safe and effective health care foods for assisting in reducing blood glucose and improving memory function in diabetic patients, and drugs for intervening in cognitive dysfunction in diabetic patients.

[0003] Patent No. CN118271398A discloses a pig collagen-derived hypoglycemic peptide, which is prepared by extracting collagen from pigskin through ultrasonic-assisted acid method and then enzymatically hydrolyzing with pepsin. The hypoglycemic peptide can promote Min6 cells to secrete insulin and improve the absorption of glucose by pancreatic beta cells, thereby playing a hypoglycemic role. Patent No. CN111607628A prepares sea cucumber and camel blood hypoglycemic mixed peptides through protease enzymolysis, membrane separation, activated carbon decolorization, deodorization, and spray drying technology, respectively. The two are mixed in a ratio of 1-1.5:1 to prepare a sea cucumber and camel blood hypoglycemic mixed peptide. The sea cucumber and camel blood hypoglycemic mixed peptide has an IC 50 of 0.5 mg / mL for α-amylase inhibition, an IC 50 of 51 μg / mL for α-glucosidase inhibition, and an IC 5043 μg / mL, with good in vitro hypoglycemic activity. At present, the hypoglycemic active mixed peptides are mainly obtained by adding protease to hydrolyze the raw material after extracting the protein, and then by a series of separation and purification, the operation process is relatively complex, the peptide sequence is not clear, and in vitro activity evaluation is mainly used, and whether it has in vivo hypoglycemic activity is not clear. The patent with publication number CN117778511A discloses a preparation method and application of royal jelly protein hypoglycemic peptide powder, taking the protein in fresh royal jelly as raw material, three times of enzymolysis by alkaline protease, pepsin and trypsin, and freeze-drying to obtain royal jelly mixed peptide powder, using calculated enzymolysis peptide segment, combining glycosidase peptide library and LBSPepPredictor tool to identify seven high-activity peptide segments GSR, LW, IW, TW, IF, PR and CL, wherein PR shows strong α-glucosidase inhibitory activity (19.79 μmol / L). The preparation steps of the invention are complex, and the hypoglycemic active mixed peptides are obtained by three times of enzymolysis and freeze-drying, and in vitro hypoglycemic activity evaluation is mainly used, and whether it has in vivo hypoglycemic activity is not clear. The currently reported hypoglycemic peptides mainly assist in reducing blood sugar by α-glucosidase inhibitory activity, and lack of hypoglycemic peptides for regulating blood sugar, intervening and improving cognitive dysfunction of diabetes. SUMMARY

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

[0005] The hypoglycemic peptide of the present application is extracted from ham.

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

[0007] Preferably, a preparation method of a hypoglycemic peptide is as follows:

[0008] (1) A ham is weighed, ground, and then added with a phosphate buffer solution, and the supernatant is collected by centrifugation. Ethanol solution is added to the supernatant, which is then placed at 4℃ for 12 h, centrifuged, and the supernatant is collected. The supernatant is ultrafiltered by a filter membrane with a molecular weight cut-off of 10 KDa, and the filtrate is collected to obtain a hypoglycemic crude peptide of ham.

[0009] (2) The hypoglycemic crude peptide of ham obtained in step (1) is subjected to peptide purification and peptide amino acid sequence identification by liquid chromatography-mass spectrometry (LC-MS / MS), and two peptides with an amino acid sequence of Asp-Ser-Val-Phe (DSVF) and Thr-Leu-Ala-Glu (TLAE) are obtained.

[0010] Preferably, the mass-volume ratio of the ham to the phosphate buffer in step (1) is (1-3):(2-20) 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℃ for 20 min.

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

[0013] Preferably, in step (2), Q-Exactive LC-MS / MS is used for purification and analysis, and PeaksStudio software is used for identifying the amino acid sequence of the hypoglycemic peptide. Agilent Eclipse Plus C18 column (2.1mm*150mm, 3.5μm) is used for separation and purification, mobile phase A is water:formic acid=1000:1(V / V), mobile phase B is acetonitrile:formic acid=1000:1(V / V), elution with 5% B phase for 0-5min, elution with 5%-20% B phase for 5-10min, elution with 20%-40% B phase for 10-25min, elution with 40%-75% B phase for 25-35min, elution with 5% B phase for 35-40min, flow rate is 0.2mL / min, column temperature is 30℃, injection volume is 5μL, injection concentration is 0.1mg / mL, detection wavelength is 220nm, mass spectrometry resolution is Full MS 35000, mass spectrometry detection is carried out in positive ion mode, mass range is 100-2000m / z, and collision energy is 10eV, 20eV and 30eV.

[0014] The third object of the present application is to provide an application of the hypoglycemic peptide in preparing a medicine for improving diabetes and cognitive dysfunction of diabetes patients.

[0015] Advantages of the present application

[0016] (1) The hypoglycemic peptide with a novel amino acid sequence can reduce blood sugar and improve cognitive dysfunction of diabetes patients, which is verified by animal experiments.

[0017] (2) The hypoglycemic peptide can be extracted from ham, and the raw material source is simple, safe and prepared by a conventional method. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Trajectory graph of the open field experiment of the DSVF intervention group of mice Figure 1 (A), center area exploration time histogram Figure 1 (B), and straight and modification frequency histogramFigure 1 (C))).

[0019] Figure 2 Trajectory graph of the open field experiment for the DSVF intervention group mice Figure 2 (A), center area exploration time histogram (B), and the number of rearing and grooming histogram (C). Figure 2 Figure 2

[0020] Figure 3 Trajectory graph of the novel object recognition experiment for the DSVF intervention group mice (A) and the NOI recognition index histogram (B). Figure 3 Figure 3 Trajectory graph of the novel object recognition experiment for the TLAE intervention group mice (A) and the NOI recognition index histogram (B).

[0021] Figure 4 Figure 4 Trajectory graph of the novel object recognition experiment for the TLAE intervention group mice (A) and the NOI recognition index histogram (B). Figure 4

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

[0023] Effect of TLAE on the release levels of CRH, ACTH and CORT of the HPA axis of mice. Figure 6 DETAILED DESCRIPTION The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following embodiments are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0024] Example 1

[0025] Preparation and screening of hypoglycemic peptides.

[0026] (1) 10 g of Xuanwei ham meat was minced with a mincer and then added with 40 ml of phosphate buffer solution (pH 7.4), and the supernatant was collected by centrifugation at 12000 rpm / min for 20 min at 4℃. 120 ml of 40% ethanol solution was added to the supernatant, and the mixture was placed at 4℃ for 12 h, and then centrifuged at 12000 rpm / min for 20 min at 4℃ to collect the supernatant.

[0027]

[0028] ​​​​​(2) Take 20 g of Wenshan ham, grind it with a grinder, and add 100 ml of phosphate buffer (pH 7.4). Centrifuge at 4°C at 12000 rpm / min for 20 min to collect the supernatant. Add 300 ml of 40% ethanol solution, and stand at 4°C for 12 h. Centrifuge at 4°C at 12000 rpm / min for 20 min to collect the supernatant.

[0029] (3) The supernatants collected in steps (1) and (2) are separately ultrafiltered with a filter membrane with a molecular weight cut-off of 10 KDa, and the filtrate is collected to obtain crude hypoglycemic peptides of Xuanwei ham and Wenshan ham.

[0030] (4) Purify and analyze using Q-Exactive LC-MS / MS, and identify the amino acid sequence of the hypoglycemic peptides using PeaksStudio software. Purify and separate using an Agilent Eclipse Plus C18 column (2.1 mm x 150 mm, 3.5 μm), mobile phase A is water:formic acid = 1000:1 (V / V), mobile phase B is acetonitrile:formic acid = 1000:1 (V / V), elute with 5% B phase for 0-5 min, elute with 5%-20% B phase for 5-10 min, elute with 20%-40% B phase for 10-25 min, elute with 40%-75% B phase for 25-35 min, elute with 5% B phase for 35-40 min, flow rate is 0.2 mL / min, column temperature is 30°C, sample injection amount is 5 μL, sample injection concentration is 0.1 mg / mL, detection wavelength is 220 nm. The mass resolution is Full MS 35000, and mass spectrometry is performed in positive ion mode, mass range is 100-2000 m / z, collision energy is 10 eV, 20 eV, and 30 eV. The polypeptide sequences identified from the crude hypoglycemic 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), and the polypeptide sequences identified from the crude hypoglycemic 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) The three-dimensional structure of the hypoglycemic peptide was drawn by SYBYL-X 2.1.1 software. 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 hypoglycemic peptide obtained in step (4) was subjected to molecular docking analysis with the two proteins (α-amylase and DPP-IV). The number of hydrogen bonds and the number of hydrogen bonds between the receptor and the ligand were calculated. 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) were selected as the top four (Table 1).

[0032] (6) Peptide synthesis: The four peptides selected in step (5) were entrusted to Nanjing Jiepeibio Technology Co., Ltd. for solid-phase synthesis, desalination, and purity ≥98%.

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

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

[0035] In a 2 mL centrifuge tube, 0.4 mL of sample and 0.4 mL of α-amylase (700 U / g) were added and mixed. After incubation at 37°C water bath for 10 min, 0.4 mL of soluble starch (1%) was added and incubated for another 3 min. Then 0.4 mL of DNS reagent was added and boiled for 10 min. The absorbance value was measured at 540 nm and recorded as A 样品 .

[0036] The absorbance value was measured with phosphate buffer instead of DNS and recorded as A 对照 ;

[0037] The absorbance value was measured with ultrapure water instead of sample and recorded as A 空白 ;

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

[0039] The formula for calculating the inhibition rate of α-amylase is:

[0040]

[0041] The five concentration peptide solutions were plotted against the corresponding alpha-amylase inhibition rate to draw a standard curve, and the alpha-amylase 50% inhibition rate concentration, i.e. IC50value, was obtained. 50

[0042] DPP-IV inhibition rate determination: The four peptides synthesized by solid phase synthesis in step (6) were configured into solutions with concentration gradients of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL, and sitagliptin with the same concentration was used as a positive control. All reagents in the experiment were configured with 100 mM Tris-buffer 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, and after incubation at 37°C for 1 h, 200 μL of the mixture was taken and placed in a 96-well plate, and the absorbance was measured at 405 nm.

[0044] Tris-buffer buffer was used instead of the sample as a control group;

[0045] Tris-buffer buffer was used instead of the enzyme as a color group,

[0046] Sitagliptin was used instead of the sample as a positive control.

[0047]

[0048] The five concentration peptide solutions were plotted against the corresponding DPP-IV inhibition rate to draw a standard curve, and the DPP-IV 50% inhibition rate concentration, i.e. IC50value, was obtained.

[0049] In vitro analysis of alpha-amylase and DPP-IV inhibition rate was performed (Table 2), and the most active DSVF (Asp-Ser-Val-Phe) and TLAE (Thr-Leu-Ala-Glu), i.e. the hypoglycemic peptide, was obtained.

[0050] Table 1 Docking results of hypoglycemic peptides with alpha-amylase and DPP-IV

[0051]

[0052] Table 2 Inhibition rate of hypoglycemic peptides on alpha-amylase and DPP-IV

[0053] Peptide sequence Alpha-amylase inhibition IC 50 (mg / mL) 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] Improving effect of hypoglycemic peptides on cognitive dysfunction of T2DM mice (type 2 diabetes mellitus mice) ​

[0056] (1) Establishment of T2DM mouse model: 40 male C57BL / 6J mice aged 4-5 weeks (weighing 28-20 g) were randomly divided into a normal group (Control), a diabetic 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). Throughout the experiment, the control group mice were fed with ordinary feed, while the diabetic mice were fed with a 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, 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, the mice were classified 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 model of each group of mice was successfully established, 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 subjected to gavage intervention. 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 hypoglycemic peptide were gavaged with 5 mg / kg and 10 mg / kg hypoglycemic peptide every day, respectively, 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 level in the diabetic model group was maintained between 12.40 and 13.40, indicating that the group was in a state of hyperglycemia. In the first week of oral administration of Aca and glucose-lowering peptide to T2DM mice, the fasting blood glucose levels of all intervention groups except the TL group decreased, and the fasting blood glucose levels of all intervention groups gradually decreased during the continuous oral administration. The fasting blood glucose level of the Aca group reached normal levels after 3 weeks of oral administration. After 4 weeks of oral administration, the fasting blood glucose levels of DL, DH, TL, and TH mice decreased significantly compared with those in the Model group. The fasting blood glucose levels of the DH and TH groups were 7.80 mmol / L and 7.40 mmol / L, respectively, from the first week, both reaching normal levels. The results of fasting blood glucose monitoring indicate that glucose-lowering peptides have the effect of regulating the fasting blood glucose level in T2DM mice.

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

[0059]

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

[0061] The cognitive ability of mice was evaluated by the novel object recognition test of the difference in exploration time of familiar objects and new objects. Two objects (A and B) of the same size, color and shape were placed in the open field, and the two objects were fixed. Before the experiment, the mice were placed in the open field for free exploration for 10 min, and then returned to the cage for rest. 6 h later, object B was replaced by object C with different color and shape, and the mice were placed in the same field in the same way for free exploration for 10 min. After each mouse finished exploring, the field was cleaned with 75% alcohol. The activity trajectory of the mouse was recorded using a camera.

[0062] (2) After 4 weeks, the mice were sacrificed by cervical dislocation, and 0.1 g of plasma, brain and intestinal tissue was taken and homogenized in 0.9 mL of physiological saline at low temperature. Centrifugation was performed at 9000 rpm / min for 15 min, and the supernatant was used to determine the levels of CRH (corticotropin releasing hormone), ACTH (adrenocorticotropic hormone) and CORT (corticosterone) in serum, brain tissue and intestinal tissue according to the kit instructions.

[0063] Results analysis

[0064] Compared with the Control group mice, the total travel distance and exploration time of the Model group mice in the center area were significantly reduced ( Figure 1 (B)), and the number of rears and modifications was significantly increased ( Figure 1 (C)), the exploration time of the center area of the Aca group, DL and DH intervention groups was significantly increased ( Figure 1 (B)), and the number of rears and modifications was significantly decreased ( Figure 1 (C)). From the mouse activity trajectory graph, it can be directly reflected that compared with the Model group, the free exploration ability of the Aca group, DL and DH intervention group mice was increased ( Figure 1 (A)).

[0065] As Figure 2 showed, the activity distance and residence time of the Model group mice in the center area were significantly reduced ( Figure 2 (B)), and the number of rears and modifications was significantly increased ( Figure 2 (C), which was significantly different from the Control group mice. After Aca, TL and TH intervention, the residence time of the mice in the center area (Aca group was 16.27 s, TL group was 16.49 s, TH group was 19.09 s) was significantly increased compared with the Model group (9.06 s) Figure 2(B)) and the number of standing and modification times decreased from 29.00 times in the Model group to 20.00 times in the Aca group, 19.00 times in the TL group, and 19.00 times in the TH group, with significant differences Figure 2 (C)). As can be seen from the mouse activity trajectory graph, compared with the Model group, the free exploration ability of mice in the Aca group, the TL and TH intervention groups increased Figure 2 (A)). The results showed that DSVF and TLAE can improve the free exploration ability of T2DM mice.

[0066] The results of the novel object recognition experiment of mice in the DSVF intervention group are shown in Figure 3 Compared with the Control group, the novelty discrimination index (NOI) of the Model group decreased by nearly half Figure 3 (B)), indicating that the mice in the Model group could not distinguish between new objects and familiar objects. The NOI of the Aca group, the DL and DH intervention groups increased significantly Figure 3 (B)).

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

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

[0069] The results of the effect of TLAE on the levels of CRH, ACTH and CORT in the HPA axis of T2DM mice are shown in Figure 6 Compared with the Control group, the levels of CRH, ACTH and CORT in the serum, brain tissue and intestinal tissue of mice in the Model group 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, and there was a significant difference with the Model group. The results showed that DSVF and TLAE can improve the dysfunction of the HPA axis of T2DM mice.

[0070] In summary, the hypoglycemic peptide of the present embodiment has the potential to be developed as a drug for assisting in reducing blood sugar, improving memory function of diabetic patients, and intervening cognitive dysfunction of diabetic patients.

Claims

1. Use of a glucose-lowering peptide in the preparation of a drug for improving diabetes, wherein the amino acid sequence of the glucose-lowering peptide is Asp-Ser-Val-Phe or Thr-Leu-Ala-Glu.

2. Use of a glucose-lowering peptide in the preparation of a drug for improving diabetic cognitive dysfunction, wherein the amino acid sequence of the glucose-lowering peptide is Asp-Ser-Val-Phe or Thr-Leu-Ala-Glu.

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