Preparation method of walnut hypoglycemic peptide

Abstract

The application discloses a preparation method of a walnut blood sugar reducing peptide, and the method comprises the following steps: step one, preparing defatted walnut powder; step two, extracting walnut protein; step three, preparing walnut meal crude peptide; step four, DPP-IV enzyme and alpha-glucosidase inhibiting peptide separation; and step five, DPP-IV enzyme and alpha-glucosidase inhibiting peptide screening. The method prepares a peptide LPWR which has relatively high DPP-IV enzyme and alpha-glucosidase inhibiting activities, the molecular weight of the peptide is 571.34 Da, and IC 50 values are 278.6 muM and 353.0 muM respectively. The LPWR is closely combined with the DPP-IV enzyme and the alpha-glucosidase mainly through hydrogen bonds and van der Waals forces. The walnut blood sugar reducing peptide has a significant improvement effect on glucose metabolism of normal mice. Therefore, the walnut blood sugar reducing peptide provides a theoretical basis for type II diabetes treatment and improvement.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of a blood sugar lowering peptide, and in particular to a method for preparing a blood sugar lowering peptide from walnuts. BACKGROUND

[0002] Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar, mainly divided into type I, type II (T2D) and gestational diabetes. The proportion of patients with T2D is more than 90% of the total number of diabetic patients, which is a non-communicable endocrine disease caused by impaired or absent insulin signaling. According to the data of International Diabetes Federation in 2021, about 540 million adults (20-79 years old) in the world have diabetes, and the proportion of patients in China accounts for 26%. Persistent high blood sugar and long-term metabolic disorders can cause damage and dysfunction or failure of systemic organs, especially kidney, eye, nervous system and cardiovascular system. Therefore, it is urgent to develop safe and effective active substances to effectively intervene T2D.

[0003] Dipeptidyl peptidase IV (DPP-IV) is a multifunctional transmembrane glycoprotein widely distributed in human organs and tissues. DPP-IV mainly acts on glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), destroys its segment, so that it loses the function of stimulating insulin secretion, thereby losing the ability to maintain postprandial blood glucose balance. Therefore, inhibition of DPP-IV activity has been proven to be an effective and safe treatment for T2D. Alpha-glucosidase is mainly located in the brush border cells of the small intestine mucosa, and is a target enzyme for regulating postprandial blood glucose. Alpha-glucosidase is crucial in catalyzing the last step of carbohydrate digestion, and inhibiting its activity can interfere with the digestion of carbohydrates in the diet to produce glucose, thereby achieving good control of postprandial blood glucose.

[0004] Walnut is one of the four major dry fruits in the world and has high nutritional value. Walnut kernel, as the main edible part of walnut, contains nutrients such as protein, lipid, carbohydrate, cellulose, vitamins and minerals. Walnut protein is a high-quality plant protein, containing 18 kinds of amino acids, and the 8 kinds of essential amino acids for human body are complete and balanced in proportion. Walnut cake meal, as a byproduct after walnut oil extraction, is usually used as feed or discarded in large quantities, which will cause unnecessary resource waste and environmental pollution. Therefore, it is the focus of walnut resource utilization to deeply process walnut meal protein and improve the comprehensive utilization efficiency of walnut resources. Therefore, it is of great significance to develop a method for preparing blood sugar lowering peptides based on walnuts. SUMMARY

[0005] The application aims to provide a preparation method of walnut hypoglycemic peptide, which prepares a peptide LPWR with high DPP-IV enzyme and alpha-glucosidase inhibitory activity, and the walnut hypoglycemic peptide has a significant improvement effect on glucose metabolism of normal mice.

[0006] The application aims to achieve the above-mentioned purpose through the following technical scheme.

[0007] The application aims to achieve the above-mentioned purpose through the following technical scheme.

[0008] Step one, preparing defatted walnut powder: after shelling and crushing, the walnut is mixed with n-hexane solution in a 37℃ shaking bed for defatting, the defatted walnut meal is dried, crushed and sieved to obtain the defatted walnut powder;

[0009] Step two, extracting walnut protein: the defatted walnut powder is mixed with distilled water at a ratio of 1:8-12 (w / v), the solution pH is adjusted to 9.5-10.5 by NaOH, and the solution is extracted by a magnetic stirrer at 50-60℃ for 1-2h, and then the supernatant is obtained by centrifugation; the supernatant pH is adjusted to 4.0-4.5 by HCl solution, and the solution is stirred for 1-2h, and then the lower layer precipitate is obtained by centrifugation, and the precipitate is dialyzed, desalted and freeze-dried to obtain the walnut protein;

[0010] Step three, preparing walnut meal crude peptide: the walnut protein is dissolved in distilled water at 2-4% (w / v) to form a walnut protein solution and treated in a 90-100℃ water bath for 10-30min, then the temperature of the walnut protein solution is adjusted to 35-40℃, the pH is adjusted to 7.8-8.5, and 6000U / g of trypsin is added for reaction, and then the solution is placed in a boiling water bath for 10-15min, the supernatant is obtained by centrifugation and freeze-dried to obtain the walnut meal crude peptide;

[0011] Step four, DPP-IV enzyme and alpha-glucosidase inhibitory peptide separation: the walnut crude peptide is preliminarily separated and purified by a DEAE-52 cellulose ion exchange column, and then eluted by 0.02mol / L PBS and 0.1, 0.2, 0.3 and 0.4mol / L NaCl solution in sequence, and the collected sample solution is dialyzed, desalted and freeze-dried, and the inhibitory activity of different components on DPP-IV enzyme and alpha-glucosidase is determined;

[0012] Step five, DPP-IV enzyme and alpha-glucosidase inhibitory peptide screening: the best separation component with the best inhibitory activity screened in step four is prepared into a sample solution at 6-10mg / mL, and then further separated and purified by a Sephadex-G25 dextran gel column, and then eluted by distilled water, and then the eluted components are enriched and freeze-dried to obtain the walnut hypoglycemic peptide.

[0013] Compared with the prior art, the present application has the following advantages:

[0014] The hypoglycemic peptide prepared in the present application is Leu-Pro-Trp-Arg (LPWR), with a molecular weight of 571.34 Da, and has high DPP-IV enzyme and alpha-glucosidase inhibitory activities, with IC 50 values of 278.6 μM and 353.0 μM, respectively. LPWR is closely combined with DPP-IV enzyme and alpha-glucosidase mainly through hydrogen bonds and van der Waals forces. After normal glucose loading (2 g / kg body weight) of mice, 40 mg / kg of LPWR can reduce the postprandial blood glucose level of mice compared with the control group. Moreover, the contents of insulin, GLP-1 and GIP in the serum of mice treated with LPWR are higher than those of the control group. The hypoglycemic mechanism of the walnut peptide disclosed in the present application can provide a theoretical basis and reference for the development of high-efficiency, sensitive and safe hypoglycemic dietary supplements in the future, and can also strengthen the high-value utilization of walnuts. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A: Influence of hydrolysis time on the degree of hydrolysis, B and C: Inhibitory activities of the walnut protein trypsin hydrolysate on DPP-IV enzyme and alpha-glucosidase.

[0016] Figure 2 A: DEAE-52 chromatography separation diagram, B and C: Inhibitory rates of the DEAE-52 separated components on DPP-IV enzyme and alpha-glucosidase.

[0017] Figure 3 A: Sephadex G-25 chromatography separation diagram, B and C: Inhibitory rates of the Sephadex G-25 separated components on DPP-IV enzyme and alpha-glucosidase.

[0018] Figure 4 H3-2 component total ion flow chromatogram based on LC-MS / MS analysis in the present application.

[0019] Figure 5 Mass spectrum diagram of Leu-Pro-Trp-Arg (LPWR) in the present application.

[0020] Figure 6Molecular docking results of LPWR with DPP-IV enzyme and alpha-glucosidase in the application, A: molecular docking results of LPWR with DPP-IV enzyme, B: molecular docking results of LPWR with alpha-glucosidase.

[0021] Figure 7 Effect of LPWR on blood glucose of normal mice in the application.

[0022] Figure 8 Effect of LPWR on insulin, GLP-1 and GIP of normal mice in the application, A: effect of LPWR on insulin of normal mice, B: effect of LPWR on GLP-1, C: effect of LPWR on GIP. DETAILED DESCRIPTION

[0023] The technical solutions of the application are further described below with reference to the drawings, but are not limited thereto, and any modification or equivalent replacement to the technical solutions of the application without departing from the spirit and scope of the technical solutions of the application shall be covered in the protection scope of the application.

[0024] The application provides a preparation method of walnut blood glucose-lowering peptide, and the method comprises the following steps:

[0025] Step one: after shelling and crushing the walnut, the walnut is mixed with n-hexane solution in a 37℃ shaking bed for defatting, the defatted walnut meal is dried, crushed and sieved to obtain defatted walnut powder, wherein: the walnut is mixed with n-hexane at a ratio of 1:3-5 (w / v).

[0026] Step two: the defatted walnut powder is mixed with distilled water at a ratio of 1:8-12 (w / v), the pH of the solution is adjusted to 9.5-10.5 by using NaOH, and then the solution is extracted by using a magnetic stirrer at 50-60℃ for 1-2h, and the supernatant is obtained by centrifugation; the pH of the supernatant is adjusted to 4.0-4.5 by using an HCl solution, and then the supernatant is stirred for 1-2h, and the lower layer of the supernatant is obtained by centrifugation, and then the lower layer is dialyzed, desalted and freeze-dried to obtain walnut protein.

[0027] Step three: the walnut protein is dissolved in distilled water at a ratio of 2-4% (w / v) to form a walnut protein solution, and then the walnut protein solution is treated in a water bath at 90-100℃ for 10-30min, then the temperature of the walnut protein solution is adjusted to 35-40℃, the pH is adjusted to 7.8-8.5, and then 6000U / g of trypsin is added for reaction, and then the walnut protein solution is placed in a boiling water bath for 10-15min, and then the supernatant is obtained by centrifugation and freeze-dried to obtain walnut crude peptide.

[0028] Step four: the walnut crude peptide was separated and purified by DEAE-52 cellulose ion exchange column, and was eluted by 0.02 mol / L PBS and 0.1, 0.2, 0.3 and 0.4 mol / L NaCl solution in sequence. The collected sample solution was dialyzed to remove salt and then freeze-dried. The inhibitory activities of different components on DPP-IV and α-glucosidase were determined. The concentration of walnut crude peptide was 8-10 mg / mL, the loading amount was 6-10 mL, and the flow rate was set to 2 mL / min.

[0029] Step five: the best separation component with the best inhibitory activity screened in step four was prepared into a polypeptide solution with a concentration of 10 mg / mL, and the molecular weight distribution was determined by gel permeation chromatography (PL-GPC50). The type and size of the chromatographic column were aquagel-OH Mixed-M and 7.5 mm x 300 mm, respectively. Water + 0.2 M NaNO3+ 0.01 M NaH2PO4(pH = 7) was used as the mobile phase, and the flow rate was 1.0 mL / min. 1560, 2250, 3480, 4320, 6400, 21900, 400, 107000, 348000 and 1046000 Da dextran were used as standard products.

[0030] Step six: the best separation component with the best inhibitory activity screened in step four was prepared into a sample solution with a concentration of 6-10 mg / mL, and was further separated and purified by Sephadex-G25 dextran gel column. Distilled water was used for elution, and the elution rate was 0.5 mL / min. After the elution components were enriched and freeze-dried, the inhibitory activities of different components on DPP-IV and α-glucosidase were determined.

[0031] Step seven: The polypeptide components with the best inhibitory activity screened in step six were identified by liquid chromatography tandem mass spectrometry (LC-MS / MS). The sample was determined for fragment structure and sequence by an EASY-nLC 1200 system (Thermo Fisher Scientific) of an electrospray-combination ion trap mass spectrometer, an RP-C18 filler packed pre-column (150 μm x 2 cm), and an RP-C18 separation capillary column (100 μm x 180 mm). The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile solution (80% acetonitrile). At 0 min, the volume ratio of B phase was 4%. At 3 min, the volume ratio of B phase was increased to 8%. The volume ratio of B phase was increased from 8% to 28% at 89 min. The volume ratio of B phase was maintained at 95% at 110-120 min. The flow rate was 600 nL / min. After the sample was separated by high performance liquid chromatography, mass spectrometry analysis was performed in positive ion scanning mode, and the parent ion scanning range was 100-1500 (m / z). The resolution and automatic gain control target (AGC target) of MS1 were set to 70000 and 3 x 10 6 , respectively. The maximum injection time was 100 ms. In MS2, the resolution was 17500, and the AGC target was 1 x 10 5 . The maximum injection time was 50 ms.

[0032] Step eight: The inhibitory mechanism of the peptides identified in step seven on DPP-IV enzyme and a-glucosidase was revealed by molecular docking. The 2D and 3D structures of the polypeptides were drawn using ChemDraw 20.0 and Chem3D 20.0, respectively. The DPP-IV crystal structure (PDB ID: 1J2E, Resolution: ) was obtained from the protein database (https: / / www.rcsb.org / ). Molecular docking was performed using AutoDock Vina. Finally, the docking results were visualized using PyMOL and Discovery Studio 2019 Client.

[0033] Example 1:

[0034] The present embodiment provides a preparation method of walnut hypoglycemic peptides, which comprises the following steps:

[0035] (1) The walnut was shelled and crushed, and then mixed with n-hexane solution at a ratio of 1:5 (w / v) for defatting in a 37°C shaking bed. The n-hexane was replaced every 1 h. The defatted walnut meal was air-dried, crushed, and then sieved (60 mesh).

[0036] (2) Defatted walnut powder was mixed with distilled water at a ratio of 1:10 (w / v), and the pH of the solution was adjusted to 9 using NaOH. After 1.5 h of extraction at 50°C using a magnetic stirrer, the supernatant was obtained by centrifugation. The pH of the supernatant was adjusted to 4.0 using an HC1 solution, and the mixture was stirred thoroughly for 1.5 h. After centrifugation, the lower precipitate was obtained. After dialysis and desalination, the precipitate was freeze-dried to obtain walnut protein.

[0037] (3) Walnut protein was dissolved in distilled water at a concentration of 2% to form a walnut protein solution, which was then treated in a water bath at 90°C for 10 min. The temperature of the walnut protein solution was then adjusted to 40°C, and the pH was adjusted to 8. Then, 6000 U / g of trypsin was added for reaction. After the enzymatic reaction, the solution was placed in a boiling water bath for 10 min. The supernatant was obtained by centrifugation and freeze-dried to obtain walnut crude peptides.

[0038] As shown in Figure 1 , the hydrolysis rate decreased and tended to be stable as the hydrolysis proceeded. The degree of hydrolysis was 14.02 ± 0.54% after 5 h of trypsin hydrolysis. The inhibition rates of the trypsin hydrolysate on DPP-Ⅳ and α-glucosidase were 44.73 ± 1.60% and 34.10 ± 1.60%, respectively.

[0039] (4) A chromatography column with a size of 2.6 cm x 30 cm was selected, and the column was equilibrated with 0.01 mol / L PBS buffer. A walnut peptide solution with a concentration of 10 mg / mL was prepared and slowly loaded onto the column. The flow rate was set to 2 mL / min, and the column was eluted with 0.02 mol / L PBS and 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solutions, respectively. The changes in the elution profile were observed under ultraviolet light at a wavelength of 280 nm. The samples were collected according to the elution curve, and the collected sample solutions were dialyzed, desalted, and freeze-dried. The component with the highest inhibition rate was selected for gel chromatography purification.

[0040] As shown in Figure 2 , components H1 (0.02 mol / L PBS), H2 (0.1 mol / L NaCl), H3 (0.2 mol / L NaCl), and H5 (0.4 mol / L NaCl) all showed inhibition activity on DPP-Ⅳ, among which component H3 had the highest inhibition rate on DPP-Ⅳ, which was 27.03 ± 1.74%. Components H1, H2, H3, and H5 also had α-glucosidase inhibition activity, among which component H3 had the highest inhibition rate on α-glucosidase, which was 16.88 ± 1.64%.

[0041] (5) The best DEAE-52 cellulose column separation component with inhibitory activity was allocated as a polypeptide solution of 10 mg / mL. It was used for standby after filtration by microporous filter membrane (0.22 μM). The molecular weight distribution was determined by using gel permeation chromatography (USA, PL-GPC50). The detector was differential refractive detector. The type and size of the chromatographic column were aquagel-OH Mixed-M and 7.5 mm x 300 mm, respectively. The mobile phase was water + 0.2 M NaNO3+ 0.01 M NaH2PO4(pH = 7) with a flow rate of 1.0 mL / min. The standard samples were dextran with molecular weights of 1560, 2250, 3480, 4320, 6400, 21900, 400, 107000, 348000 and 1046000 Da, respectively.

[0042] (6) The best separation component with inhibitory activity screened in step (4) was allocated as a sample solution of 10 mg / mL. The sample solution was filtered by 0.22 μm filter membrane to remove impurities, and then slowly added to a Sephadex-G25 gel column along the side wall. The sample loading amount was 4 mL. Distilled water was used for elution with an elution rate of 0.5 mL / min. The ultraviolet absorption peak at 220 nm was measured, and the sample was collected by the change trend of the absorption peak graph. After the elution components were enriched and freeze-dried, the inhibitory activities of different components on DPP-IV enzyme and α-glucosidase were determined.

[0043] As shown in Figure 3 , two obvious elution peaks (H3-1 and H3-2) were obtained after H3 was separated in the present embodiment. The DPP-IV inhibition rates of H3-1 and H3-2 were 17.81 ± 1.33% and 24.10 ± 1.59%, respectively, and the α-glucosidase inhibition rates were 10.97 ± 0.26% and 14.77 ± 0.45%, respectively.

[0044] (7) An α-glucosidase solution (0.5 U / mL), a p-nitrophenyl-α-D-glucopyranoside solution (4 mmol / L, PNPG), a walnut polypeptide solution (0.5 mg / mL) and a Na2CO3 solution (0.2 mol / L) were prepared by using PBS buffer (0.2 mol / L, pH = 6.8). The PBS buffer, PNPG and sample solution were sequentially added to a 96-well enzyme-labeled plate, and incubated at 37°C for 10 min. After removal, the α-glucosidase solution was added, mixed uniformly, and incubated at 37°C for 30 min. Then, the reaction was terminated by immediately adding the Na2CO3 solution. The absorbance of the reaction solution was measured at a wavelength of 405 nm by using an enzyme-labeled instrument. Acarbose was used as a positive control in the present experiment, and each group had 3 parallel samples. The experiment was performed 3 times.

[0045] The calculation method of the inhibition rate of the polypeptide on α-glucosidase was as follows:

[0046]

[0047] wherein A S is the absorbance value of the sample group; A SB is the absorbance value of the sample blank group; A C is the absorbance value of the control group; A B is the absorbance value of the blank group.

[0048] (8) The degree of hydrolysis of walnut protein was determined by the pH-stat method. In the hydrolysis reaction, the pH of the system was maintained at the optimum pH of each enzyme, and the consumption of NaOH solution at different times (0.5, 1, 1.5, 2, 2.5, 3, 4, 5 h…) was recorded.

[0049]

[0050] wherein V and Nv are the consumption volume (mL) and concentration (mol / L) of NaOH, respectively; m is the mass of protein in the substrate (g); h tot is the total number of peptide bonds in unit mass of substrate protein (mmol / g), the h tot of walnut protein is 7.8 mmol / g; and a is the dissociation degree of amino acids, and the pK here is taken as the average isoelectric point of amino acids, which is 7.0.

[0051] (9) DPP-IV solution (0.1 U / mL), Gly-Pro-p-nitroanilide (2 mmol / L), walnut polypeptide solution (0.5 mg / mL), and sodium acetate solution (1 mol / L, pH 4.0) were prepared with Tris-HCl buffer (100 mmol / L, pH = 8.0). Under light shielding conditions, 30 μL of Gly-Pro-p-nitroanilide and 25 μL of sample solution were sequentially added to a 96-well enzyme-labeled plate, which was incubated at 37°C for 10 min. Then, 30 μL of DPP-IV solution was added to start the reaction, which was incubated at 37°C for 1 h. After the reaction was terminated by adding sodium acetate solution, the absorbance was measured at a wavelength of 405 nm.

[0052] The calculation method of the inhibition rate of polypeptide on DPP-IV enzyme is as follows:

[0053]

[0054] wherein A S is the absorbance value of the sample group; A SB is the absorbance value of the sample blank group; A C is the absorbance value of the control group; A B is the absorbance value of the blank group.

[0055] (10) The polypeptide components with the best inhibitory activity screened in step (6) are identified using liquid chromatography tandem mass spectrometry (LC-MS / MS). The fragment structure and sequence of the sample are determined by an EASY-nLC 1200 system (Thermo Fisher Scientific) of an electrospray-combination ion trap mass spectrometer, an RP-C18 packing pre-column (150 μm x 2 cm), and an RP-C18 separation capillary column (100 μm x 180 mm). The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid acetonitrile solution (80% acetonitrile). At 0 min, the volume ratio of B phase is 4%. At 3 min, the volume ratio of B phase is increased to 8%. The volume ratio of B phase is increased from 8% to 28% at 89 min. The volume ratio of B phase is maintained at 95% at 110-120 min. The flow rate is 600 nL / min. After the sample is separated by high performance liquid chromatography, mass spectrometry analysis is performed in positive ion scanning mode, and the parent ion scanning range is 100-1500 (m / z). The resolution and automatic gain control target (AGC target) of MS1 are set to 70000 and 3 x 105, respectively. The maximum injection time is 100 ms. In MS2, the resolution is 17500, and the AGC target is 1 x 105. The maximum injection time is 50 ms. 6 5

[0056] As shown in Figure 4 and Figure 5 , the molecular weight of LPWR is 571.34 Da, and LPWR has high DPP-IV enzyme and α-glucosidase inhibitory activity, with IC 50 values of 278.6 μM and 353.0 μM, respectively.

[0057] (11) The inhibitory mechanism of the peptide identified in step (10) on DPP-IV enzyme and α-glucosidase is revealed using molecular docking. The 2D and 3D structures of the polypeptide are drawn using ChemDraw 20.0 and Chem3D 20.0, respectively. The DPP-IV crystal structure (PDB ID: 1J2E, Resolution: ) is obtained from the protein database (https: / / www.rcsb.org / ). Molecular docking is performed using AutoDock Vina. Finally, the docking results are visualized using PyMOL and Discovery Studio 2019 Client.

[0058] LPWR mainly interacts with DPP-IV enzyme ( Figure 6 A) and α-glucosidase ( Figure 6 ​​B) tightly bound, with binding energies of -4.81 kcal / mol and -6.72 kcal / mol, respectively.

[0059] (12) 48 mice were divided into two groups, A and B. Group A was divided into a control group and an experimental group, with 4 mice in each group, and group B was divided into a control group and an experimental group, with 20 mice in each group. The mice were fasted for 12 hours but not water. The control group was gavaged with normal saline (0.2 mL), and the experimental group was gavaged with 0.2 mL of 40 mg / kg LPWR. After 30 minutes, 30% (w / v) glucose solution (2 g / kg body weight) was gavaged. The mice in group A were tail-cut at -30 min, 0 min, 30 min, 60 min, 90 min and 120 min, and blood glucose was measured using a Yuyue blood glucose meter and blood glucose test strips. The oral glucose tolerance curve was drawn based on the blood glucose values, and the area under the glucose tolerance curve (AUC 0-120 Blood was collected from the orbits of four mice in Group B at 0, 30, 60, 90, and 120 minutes. Serum was separated after centrifugation at 3,000 g at 4°C for 10 minutes. Serum levels of insulin, GLP-1, and GIP were determined using mouse ELISA kits (Shanghai Lengton Biotechnology Co., Ltd., Shanghai, China).

[0060] The experimental results showed that the postprandial blood glucose level of mice treated with LPWR in this embodiment was lower than that of the control group ( Figure 7 ), and serum insulin ( Figure 8 A), GLP-1 ( Figure 8 B) and GIP( Figure 8 C) content was higher than that in the control group.

[0061] Example 2

[0062] This embodiment provides a method for preparing walnut hypoglycemic peptide, which comprises the following steps:

[0063] (1) After shelling and crushing the walnuts, mix with n-hexane solution at a ratio of 1:5 (w / v) and defatted in a shaker at 37°C. Replace the n-hexane every hour. Air-dry the defatted walnut meal, crush it, and sieve it (60 mesh).

[0064] (2) Defatted walnut powder was mixed with distilled water at a ratio of 1:10 (w / v), and the pH of the solution was adjusted to 10 using NaOH. After extraction at 50°C for 1.5 h using a magnetic stirrer, the supernatant was centrifuged and the pH of the supernatant was collected. The pH of the supernatant was adjusted to 4.5 using HCl solution and stirred for 1.5 h. After centrifugation, the precipitate was removed. The precipitate was dialyzed for desalination and then freeze-dried to obtain walnut protein.

[0065] (3) Walnut protein was dissolved in distilled water to form a walnut protein solution at a concentration of 3%, and then treated in a 90°C water bath for 10 min. The temperature of the walnut protein solution was then adjusted to 35°C, and the pH was adjusted to 8.5. Then, 6000 U / g of trypsin was added for reaction. After the enzymatic reaction was completed, the solution was placed in a boiling water bath for 10 min. The supernatant was obtained by centrifugation and freeze-dried to obtain walnut meal crude peptides.

[0066] As shown in Figure 1 , the hydrolysis rate slowed down and tended to be stable as the hydrolysis proceeded. The degree of hydrolysis was 13.09±0.32% after trypsin hydrolysis for 5 h. The inhibition rates of the trypsin hydrolysate on DPP-Ⅳ and α-glucosidase were 43.97±0.98% and 33.1±0.69%, respectively.

[0067] (4) A chromatography column with a size of 2.6 cm x 30 cm was selected, and the column was equilibrated with 0.01 mol / L PBS buffer. A walnut polypeptide solution with a concentration of 10 mg / mL was prepared at a volume of 10 mL and slowly loaded onto the column. The flow rate was set to 2 mL / min, and the column was eluted with 0.02 mol / L PBS and 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solutions, respectively. The changes in the elution profile were observed under ultraviolet light at a wavelength of 280 nm. The samples were collected according to the elution curve, and the collected sample solutions were dialyzed, desalted, and freeze-dried. The component with the highest inhibition rate was selected for gel chromatography purification.

[0068] As shown in Figure 2 , components H1 (0.02 mol / L PBS), H2 (0.1 mol / L NaCl), H3 (0.2 mol / L NaCl), and H5 (0.4 mol / L NaCl) all showed DPP-Ⅳ inhibition activity in this example, with the highest inhibition rate of 25.70±0.68% for component H3. Components H1, H2, H3, and H5 had α-glucosidase inhibition activity, with the highest inhibition rate of 16.21±0.82% for component H3.

[0069] (5) The best DEAE-52 cellulose column separation component with inhibitory activity was allocated as a polypeptide solution of 10 mg / mL. It was used for standby after filtration by microporous filter membrane (0.22 μM). The molecular weight distribution was determined by using gel permeation chromatography (USA, PL-GPC50). The detector was differential refractive detector. The type and size of the chromatographic column were aquagel-OH Mixed-M and 7.5 mm x 300 mm respectively. The mobile phase was water + 0.2 M NaNO3+ 0.01 M NaH2PO4(pH = 7) and the flow rate was 1.0 mL / min. The standard samples were dextran of 1560, 2250, 3480, 4320, 6400, 21900, 400, 107000, 348000 and 1046000 Da respectively.

[0070] (6) The best separation component with inhibitory activity screened in step (4) was allocated as a sample solution of 10 mg / mL. The sample solution was filtered by 0.22 μm filter membrane to remove impurities and then slowly added to Sephadex-G25 gel column along the side wall. The sample loading amount was 4 mL. Distilled water was used for elution and the elution rate was 0.5 mL / min. The ultraviolet absorption peak at 220 nm was measured and the sample was collected according to the change trend of the absorption peak graph. After the elution components were enriched and freeze-dried, the inhibitory activities of different components on DPP-IV enzyme and α-glucosidase were determined.

[0071] As shown in Figure 3 In this embodiment, two obvious elution peaks (H3-1 and H3-2) were obtained after H3 was separated. The DPP-IV inhibition rates of H3-1 and H3-2 were 14.81 ± 0.79% and 23.43 ± 1.06% respectively, and the α-glucosidase inhibition rates were 9.97 ± 0.33% and 13.43 ± 0.82% respectively.

[0072] (7) An α-glucosidase solution (0.5 U / mL), a p-nitrophenyl-α-D-glucopyranoside solution (4 mmol / L, PNPG), a walnut polypeptide solution (0.5 mg / mL) and a Na2CO3 solution (0.2 mol / L) were prepared by using PBS buffer (0.2 mol / L, pH = 6.8). The PBS buffer, PNPG and sample solution were sequentially added to a 96-well enzyme-labeled plate, which was incubated at 37°C for 10 min. After removal, the α-glucosidase solution was added and mixed uniformly, which was incubated at 37°C for 30 min. Then, the reaction was terminated by adding the Na2CO3 solution immediately. The absorbance of the reaction solution was measured by using an enzyme-labeled instrument at a wavelength of 405 nm. Acarbose was used as a positive control in this experiment, each group had 3 parallels, and the experiment was performed 3 times.

[0073] (8) The degree of hydrolysis of walnut protein was determined by the pH-stat method. In the hydrolysis reaction, the pH of the system was maintained at the optimum pH of each enzyme, and the consumption of NaOH solution at different times (0.5, 1, 1.5, 2, 2.5, 3, 4, 5 h…) was recorded.

[0074] (9) DPP-IV solution (0.1 U / mL), Gly-Pro-p-nitroanilide (2 mmol / L), walnut polypeptide solution (0.5 mg / mL), and sodium acetate solution (1 mol / L, pH 4.0) were prepared with Tris-HCl buffer (100 mmol / L, pH = 8.0). Under light-proof conditions, 30 μL of Gly-Pro-p-nitroanilide and 25 μL of sample solution were sequentially added to a 96-well enzyme plate, which was incubated at 37°C for 10 min. Then, 30 μL of DPP-IV solution was added to start the reaction, which was incubated at 37°C for 1 h. After completion, the reaction was terminated by adding sodium acetate solution, and the absorbance was measured at a wavelength of 405 nm.

[0075] (10) The polypeptide component with the best inhibitory activity screened in step (6) was identified by liquid chromatography tandem mass spectrometry (LC-MS / MS). The fragment structure and sequence of the sample were determined by the EASY-nLC1200 system (Thermo Fisher Scientific) of the electrospray-combination ion trap mass spectrometer, RP-C18 packing pre-column (150 μm x 2 cm), and RP-C18 separation capillary column (100 μm x 180 mm). The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile solution (80% acetonitrile). At 0 min, the volume ratio of B phase was 4%. At 3 min, the volume ratio of B phase was increased to 8%. The volume ratio of B phase was increased from 8% to 28% at 89 min. The volume ratio of B phase was maintained at 95% from 110 to 120 min. The flow rate was 600 nL / min. After the sample was separated by high performance liquid chromatography, mass spectrometry analysis was performed, and the positive ion scanning mode was used, with the parent ion scanning range being 100-1500 (m / z). The resolution and automatic gain control target (AGC target) of MS1 were set to 70000 and 3 x 105, respectively. The maximum injection time was 100 ms. In MS2, the resolution was 17500, and the AGC target was 1 x 105. The maximum injection time was 50 ms. 6 5

[0076] As shown in Figure 4 and Figure 5 , the molecular weight of LPWR is 571.34 Da, and LPWR has high DPP-IV enzyme and α-glucosidase inhibitory activity, with IC 50 ​​The values are 278.6 μM and 353.0 μM, respectively.

[0077] (11) The inhibition mechanism of the peptides identified in step (10) on DPP-IV and a-glucosidase was revealed by molecular docking. The 2D and 3D structures of the polypeptides were drawn using ChemDraw 20.0 and Chem3D 20.0, respectively. The crystal structure of DPP-IV (PDB ID: 1J2E, Resolution: 2.2 A) was obtained from the Protein Data Bank (https: / / www.rcsb.org / ). Molecular docking was performed using AutoDock Vina. Finally, the docking results were visualized using PyMOL and Discovery Studio 2019 Client.

[0078] LPWR binds tightly to DPP-IV (A) and a-glucosidase (B) mainly through hydrogen bonds and van der Waals forces, with binding energies of -4.81 kcal / mol and -6.72 kcal / mol, respectively. Figure 6 Figure 6

[0079] (12) 48 mice were divided into groups A and B. Group A was divided into a control group and an experimental group, each with 4 mice, and group B was divided into a control group and an experimental group, each with 20 mice. The mice were fasted for 12 hours without water restriction. The control group was given normal saline (0.2 mL) by gavage, and the experimental group was given 0.2 mL of LPWR with a concentration of 40 mg / kg by gavage. Thirty minutes later, the mice were given a 30% (w / v) glucose solution (2 g / kg body weight) by gavage. The mice in group A were tail-cut for blood at -30 min, 0 min, 30 min, 60 min, 90 min, and 120 min, and the blood glucose was measured using a Yuyue glucometer and blood glucose test strips. The oral glucose tolerance curve was plotted based on the blood glucose values, and the area under the curve (AUC 0-120 ) was calculated. The blood was taken from the orbital of 4 mice in group B at 0 min, 30 min, 60 min, 90 min, and 120 min. After centrifugation at 3,000 g at 4°C for 10 min, the serum was separated. The contents of insulin, GLP-1, and GIP in the mouse serum were measured using a mouse ELISA kit (Shanghai Lengton Biotechnology Co., Ltd., Shanghai, China).

[0080] The experimental results show that the postprandial blood glucose level of the mice treated with LPWR in this embodiment is lower than that of the control group ( Figure 7 ), and the contents of insulin ( Figure 8 A), GLP-1 ( Figure 8 B), and GIP ( Figure 8 C) in the serum are higher than those of the control group.​​​

Claims

1. Application of the hypoglycemic peptide Leu-Pro-Trp-Arg in the preparation of hypoglycemic drugs.

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