An oligopeptide with DPP-IV inhibitory activity and its preparation method and application

Abstract

The present invention belongs to the technical field of active polypeptides, and specifically relates to an oligopeptide with DPP-IV inhibitory activity, a preparation method and an application thereof. The inventor obtained a new active oligopeptide from the protein decomposition product obtained by the synergistic treatment of soybean meal enzyme and bacteria, and the amino acid sequence is as shown in SEQ ID NO.1. Studies have shown that the oligopeptide has a significant inhibitory effect on the DPP-IV enzyme, can increase the glucose consumption of insulin-resistant HePG2 cells, but does not affect the viability of HePG2 cells, and is suitable for use as a hypoglycemic active substance.

Description

Technical Field

[0001] The present invention belongs to the technical field of active polypeptides, and specifically relates to an oligopeptide with DPP-IV inhibitory activity, a preparation method and an application thereof. Background Art

[0002] A polypeptide is a substance formed by coupling amino acids. Its preparation methods usually include chemical synthesis (including solid phase synthesis, liquid phase synthesis, etc.), natural separation and purification (extraction and purification of natural polypeptides), protein decomposition and purification (preparation by natural protein hydrolysis and / or fermentation hydrolysis, etc.). Among them, solid phase synthesis is a mature polypeptide technology. It can use specific protecting groups to protect amino acids separately, and then couple and deprotect the amino acids one by one on the resin according to the amino acid sequence of the polypeptide, and then cut the prepared peptide segment from the resin to obtain the target polypeptide. Although small molecule active polypeptides with less than 40 amino acids can be prepared by chemical synthesis at present. However, most active polypeptides are discovered or decomposed from nature, and other polypeptides are obtained by structural modification such as amino acid modification based on natural polypeptides. Natural animal and plant proteins and polypeptides provide a rich resource library for the development of human medicines. For example, the glucagon-like peptide-1 (GLP-1) receptor agonist glucose-lowering peptides, which currently have a very high market share, were originally discovered in the venom of the Gila monster (the first GLP-1 receptor agonist, exenatide).

[0003] Common soybean meal is a byproduct obtained after soybean oil is extracted from soybeans, also known as "soybean meal", but there is also sedge meal available for use. Soybean meal is rich in protein and has been widely used in the fields of animal husbandry and fishery. It is also an important source of active peptides. For example, CN103343154A discloses an anti-fatigue bioactive peptide obtained by enzymatic separation of soybean meal; CN108220374A discloses a method for preparing an antioxidant active peptide from soybean meal; CN112979748B uses soybean meal to prepare an active peptide that has the effect of preventing chronic alcoholic liver damage. Soybean meal is treated with different enzymes or strains, and the resulting peptides vary greatly. In addition to the above-mentioned active peptides, it has been reported that several anti-cancer peptides, antihypertensive peptides, cholesterol-lowering peptides, etc. have been obtained from soybean meal (Cui Yunyun. Preparation of vasoactive small peptides from soybean meal and their purification, identification and activity research. Shanghai Ocean University, 2018; Sitanggang, Azis Boing, Monica Lesmana, Slamet Budijanto. Membrane-based preparative methods and bioactivities mapping of tempe-based peptides Food Chemistry 2020,329:127193; Alnuaimi A, Ajayi FF, Hamdi M,Mudgil P, Kamal H, Gan CY, Maqsood S. A comparative analysis of anti-lipidemic potential of soybean (Glycine max) protein hydrolysates obtainedfrom different ripening stages: Identification, and molecular interaction mechanisms of novel bioactive peptides. Food Chemistry, 2023,402:134192.). This further improves the utilization value of soybean meal, and its application areas expand from feed and food to therapeutic agents (including preventive or therapeutic drugs, dietary supplements, etc.).

[0004] However, there are few studies on hypoglycemic peptides in soybean meal. A large number of studies in animal husbandry and fishery have found that feeding soybean meal or fermented soybean meal as a base material to cattle, sheep, pigs, ducks or fish, etc., leads to increased blood sugar in animals (Zhang Shiyuan et al. Application effect of new fermented soybean meal in weaned piglet diet. Journal of Northwest Agriculture, 2009.). Soybean meal active peptides still have great development value. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide an oligopeptide with glucose metabolism regulating activity, a preparation method and an application, specifically an oligopeptide with DPP-IV inhibitory activity, a preparation method and an application. Oligopeptide, also known as small peptide or oligopeptide, refers to a compound formed by condensation of 2 to 10 amino acids.

[0006] The inventors obtained a new active hexapeptide and a new active decapeptide from the soybean meal semi-solid enzymatic hydrolysis and bacterial strain fermentation treatment products during the process of treating soybean meal with semi-solid enzymatic hydrolysis and bacterial strain fermentation. Both peptides have a good inhibitory effect on dipeptidyl peptidase IV (DPP-IV) enzyme and can increase the glucose consumption of insulin-resistant HepG2 cells. They are suitable as hypoglycemic preparations, especially hypoglycemic preparations for type 2 diabetes.

[0007] Therefore, one of the objects of the present invention is to provide an oligopeptide having glucose metabolism regulating activity, specifically an oligopeptide having DPP-IV inhibitory activity, wherein the oligopeptide is a hexapeptide having an amino acid sequence as shown in SEQ ID NO.1 or a decapeptide having an amino acid sequence as shown in SEQ ID NO.2.

[0008] The second object of the present invention is to provide a method for preparing the aforementioned oligopeptide, wherein the oligopeptide is prepared by any of the following methods:

[0009] Method M1: Chemical synthesis of peptides;

[0010] Method M2: soybean meal enzymatic fermentation method: soybean meal is enzymatically hydrolyzed with protease and then fermented with lactobacillus; the soybean meal is soybean meal; the protease is alkaline protease and / or neutral protease; the lactobacillus is Lactobacillus helveticus KLDS1007 (preservation number is CGMCC No. 9960) and Lactobacillus paracasei KLDS1006 (preservation number is CGMCC No.9959).

[0011] In one embodiment, the preparation method is a polypeptide solid phase synthesis method. Preferably, the polypeptide chemical synthesis method is a polypeptide solid phase synthesis method; the resin used in the polypeptide solid phase synthesis method is a 2-chlorotrityl chloride resin, the amino acid protecting group used is an Fmoc protecting group, and the amino acid condensing agent used is selected from at least one of N,N-diisopropylethylamine and HBTU.

[0012] It is well known in the art that solid phase peptide synthesis (SPPS) is a commonly used peptide synthesis method, and peptide solid phase synthesizers are now popular. The solid phase synthesis sequence is usually from the C-terminus (carboxyl end) to the N-terminus (amino end) for amino acid condensation coupling, which greatly reduces the difficulty of product purification. To prevent the occurrence of side reactions, the amino acid side chains participating in the reaction are protected. The carboxyl end is free and activated before the reaction. There are two protection strategies for solid phase synthesis: Fmoc protection strategy and tBoc protection strategy. Since Fmoc has many advantages over tBoc, Fmoc method is currently used for synthesis. There are three main types of polymer carriers (resins) used for solid phase synthesis of peptides: polystyrene-phenylene cross-linked resins, polyacrylamide, polyethylene-ethylene glycol resins and derivatives. These resins can only be directly connected to the (first) amino acid by introducing reactive groups. As a mature synthesis technology, under the premise that the amino acid sequence of the polypeptide is known, the art can refer to "Solid Phase Synthesis of Polypeptides" edited by Huang Wei De and published by Science Press in 1985, "Research and Development of Polypeptide Drugs" edited by Li Bao Qiu and published by People's Medical Publishing House in 2011, etc., for conventional selection of polypeptide solid phase synthesis resins, protecting groups, reaction solvents, cleavage reagents, etc.

[0013] In one embodiment, the preparation method is a soybean meal enzymatic fermentation method, comprising the following steps:

[0014] Step S1: semi-solid enzymatic hydrolysis of soybean meal: crushing and sieving the soybean meal, adding water and protease to obtain a semi-solid enzymatic hydrolysis product;

[0015] Step S2: fermentation of semi-solid enzymolysis product: taking the semi-solid enzymolysis product obtained in step S1, extracting with water to obtain enzymolysis soybean meal peptide extract, sterilizing the enzymolysis soybean meal peptide extract, inoculating lactic acid bacteria and fermenting to obtain soybean meal peptide fermentation liquid;

[0016] Step S3: Preliminary purification of soybean meal peptide fermentation broth: centrifuge, ultrafilter and dry the soybean meal peptide fermentation broth obtained in step S2 to obtain a peptide powder mixture.

[0017] Preferably, the amount of water added in step S1 is 0.3 mL / g soybean meal to 1 mL / g soybean meal; the amount of protease added in step S1 is ≥0.005 g protease / g soybean meal; the enzymatic hydrolysis time in step S1 is 1 h to 9 h; the lactobacillus inoculation amount in step S2 is 2% to 10%; the fermentation temperature in step S2 is 35° C. to 42° C., and the fermentation time is 18 h to 36 h.

[0018] Further preferably, the amount of protease added in step S1 is 0.005 g protease / g soybean meal to 0.03 g protease / g soybean meal; the fermentation temperature in step S2 is 36° C. to 39° C., and the fermentation time is 20 h to 28 h. The inoculation amount of the lactobacillus is 6%, and preferably, the inoculation amounts of the Lactobacillus helveticus KLDS1007 and the Lactobacillus paracasei KLDS1006 are both 3%.

[0019] The third object of the present invention is to provide the use of the aforementioned oligopeptides in the preparation of hypoglycemic active agents. Studies have shown that the aforementioned two oligopeptides have the activity of inhibiting DPP-IV enzyme and can increase the glucose consumption of insulin-resistant HepG2 cells, and are suitable for lowering blood sugar as hypoglycemic preparations, especially hypoglycemic preparations for type 2 diabetes.

[0020] Preferably, the hypoglycemic active agent is any one of an inhalation preparation, an external preparation, an injection preparation, an implant, and an oral preparation. Further preferably, the hypoglycemic active agent is an injection preparation, an implant, or an oral preparation.

[0021] The strain Lactobacillus helveticus KLDS1007 used in the invention has a deposit number of CGMCC No. 9960, a deposit unit of China National Microbiological Culture Collection Center, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit date of November 13, 2014, and a classification name of Lactobacillus helveticus; the strain Lactobacillus paracasei KLDS1006 has a deposit number of CGMCC No. 9959, a deposit unit of China National Microbiological Culture Collection Center, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit date of November 13, 2014, and a classification name of Lactobacillus paracasei.

[0022] The beneficial effects of the present invention include:

[0023] The active polypeptide for regulating sugar metabolism of the present invention has a relatively small molecular weight and is a small molecule peptide that can be absorbed as a whole by the gastrointestinal tract. It is suitable for oral administration, injection and the like, and is convenient to use.

[0024] The sugar metabolism regulating active polypeptide of the present invention can reduce blood sugar by increasing cellular glucose consumption and inhibiting DPP-IV enzyme, and is suitable for the treatment of type 2 diabetes.

[0025] The sugar metabolism regulating active polypeptide of the present invention is an active polypeptide derived from natural protein and has good safety. It is obtained by co-processing soybean meal with enzymes and bacteria, and the raw materials are easy to obtain. As a small peptide, it is prepared by a polypeptide solid phase synthesis method and has a high synthesis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The mass spectra of peptide segments 1 to 5 prepared in Example 2;

[0027] Figure 2 The mass spectra of peptide segments 6 to 10 prepared in Example 2;

[0028] Figure 3 The mass spectra of peptide segments 11 to 15 prepared in Example 2;

[0029] Figure 4 The mass spectra of peptides 16 to 20 prepared in Example 2;

[0030] Figure 5 This is the HPLC chromatogram of the target polypeptide prepared in Example 3;

[0031] Figure 6 This is the HPLC chromatogram of the target polypeptide prepared in Example 4;

[0032] Figure 7 This is a graph showing the effect of insulin on HePG2 cell viability in Example 6;

[0033] Figure 8 This is a graph showing the measurement results of the effect of insulin on glucose consumption in HePG2 cells in Example 6;

[0034] Fig. 9 This is a graph showing the effect of soybean meal peptide on HePG2 cell viability in Example 6;

[0035] Fig.10 This is a graph showing the measurement results of the effect of soybean meal peptide on glucose consumption in insulin-resistant HePG2 cells in Example 6. DETAILED DESCRIPTION

[0036] In order to make the technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail in conjunction with specific embodiments. The described embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0037] In the following examples of enzyme inhibition and cell experiments, the experimental results are expressed as mean ± standard deviation (Mean ± SD), and the statistical differences between the groups are compared by one-way analysis of variance (ANOVA), and the "Duncan" test is selected for significance analysis. Unless otherwise specified, the raw materials, reagents, consumables, and instruments used in the following examples are all commercially available products, and the detection methods used are all conventional detection methods or performed according to the instrument and equipment instructions. The soybean meal used is soybean meal (Glycinemax (L.) Merr.). Lactobacillus helveticus KLDS1007, the deposit number is CGMCC No. 9960, the deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date: November 13, 2014, the classification name: Lactobacillus helveticus. Lactobacillus paracasei KLDS1006, deposit number is CGMCC No. 9959, deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: November 13, 2014, classification name: Lactobacillus paracasei.

[0038] Example 1 Semi-solid enzymatic hydrolysis of soybean meal and mixed fermentation of lactobacillus

[0039] The semi-solid enzymatic hydrolysis conditions of soybean meal are as follows: the soybean meal is crushed through an 80-mesh sieve, water is added at a ratio of 0.5 mL / g soybean meal, and alkaline protease is added at a ratio of 0.02 g protease / g soybean meal for constant temperature oscillation enzymolysis at 40°C for 6 hours. After the semi-solid enzymatic hydrolysis product is dried and crushed in an oven, 2 g of the crushed product is added with 30 mL of water and the polypeptide is extracted in a shaker at 150 rpm and 4°C to obtain the enzymatic soybean meal peptide extract.

[0040] The enzymatically hydrolyzed soybean meal peptide extract was inactivated at 95°C for 15 min, oven dried, ground and sieved through an 80-mesh sieve, the sieved product was weighed, and constant temperature oscillation was performed at 4°C and 150 rpm for 5 h to prepare the polypeptide extract. After overnight at 4°C, the extract was sterilized in a constant temperature water bath at 95°C for 15 min, and then sterilized by ultraviolet light on a clean bench for 30 min.

[0041] Lactobacillus helveticus KLDS1007 and Lactobacillus paracasei KLDS1006 activated to the second generation by culturing in MRS liquid medium at 37°C for 18h were inoculated into the sterilized enzymatic soybean meal peptide extract at a 3% inoculation rate and fermented at 37°C for 24h. After the fermentation, the supernatant was collected after centrifugation at 3500rpm for 5min, and ultrafiltration was performed at 3500rpm for 10min using a 3kDa ultrafiltration tube to obtain a semi-solid enzymatic hydrolysis-lactobacillus fermentation mixed culture, which was freeze-dried at -80°C in a vacuum freeze-drying process for 44h to obtain a peptide powder mixture.

[0042] Example 2 Isolation, purification and primary structure identification of soybean meal peptides

[0043] The peptide powder mixture obtained in Example 1 was diluted with purified water to prepare a mixed solution with a concentration of 50 mg / mL (BCA method determination), filtered using a 3 kDa ultrafiltration device, desalted using a C18 desalting column, and then the peptides were identified using a liquid chromatography quadrupole high-resolution mass spectrometer under the following conditions: the sample was separated using a C18 reverse phase column (50 μm×15 cm, 2 μm, Thermo Fisher Scientific, San Jose, CA, USA), the injection volume was 2 μL, and the retention time was 120 min. The mobile phase eluent A was prepared by diluting a 0.1% formic acid (FA) solution with HPLC grade water, and the eluent B was prepared by diluting a 0.1% formic acid (FA) solution with 84% acetonitrile. The program flow rate was set to 300 nL / min. The concentration of eluent B was first increased from 0% to 8% (0-5 min), and then adjusted to 32% (5-105 min). The concentration of eluent B was increased from 32% to 90% (105-107 min), and the concentration of 90% was maintained for 5 min (107-112 min), and then decreased from 90% to 0% (112-120 min). The most abundant precursor ion for HCD fragmentation was dynamically selected by measuring the scan (range 350-1800 m / z) and running in positive ion mode with a resolution of 70,000. A total of 20 peptides were obtained, and their structural information is shown in Table 1. The secondary mass spectrum is shown in Figures 1 to 4 .

[0044] Table 1 Main peptides and structural information of soybean meal peptides

[0045] Peptide number Amino acid sequence (N-terminal to C-terminal) Molecular weight (Da) 1 KLGKFF 738.4416 2 AIPVNKPGRF 1097.6328 3 KIGFKF 738.4416 4 KLGFK 738.4416 5 SNLNF 740.3483 6 SQQNFLAF 924.4691 7 FISSTFAWI 1070.542 8 YDTDPFLFY 1179.5107 9 KGPDPTPGKPM 1123.5678 10 KPGFFLCSEKR 1310.6785 11 KWVLSPKYFNF 1427.7579 12 LAIPVNKPGRF 1210.7166 13 PIYSNKLGKFF 1312.7158 14 WDDGSGIPGAF 1120.481 15 EHDPDFKNPIGW 1453.6606 16 GPAGSYPPPPAG 1066.5068 17 SWDPPNPHIGIN 1345.6396 18 FEWDDGSGIPGAF 1396.5916 19 SRDPIYSNKLGKFF 1670.8754 20 SGDVWFPQPAPKDHPW 1862.8714

[0046] After inquiry, the α-glucosidase inhibitory activity of peptide No. 18 in Table 1 has been published, and peptides No. 5, 6, 8, 9, 10, 11, 12, 14, 15, 16, 17, and 20 contain dipeptide fragments that may help inhibit the DPP-IV enzyme. The hypoglycemic activity of other peptides has not been reported before. The following selects one from each of the long and short peptides according to molecular weight to investigate whether the peptide mixture and some peptide segments have glucose metabolism regulation activity. To improve research efficiency, the peptide segments were prepared by solid phase synthesis.

[0047] Example 3 Solid Phase Synthesis of Soybean Meal Active Hexapeptide KLGKFF

[0048] In this example, the oligopeptide (KLGKFF) with peptide number 1 was prepared by solid phase synthesis, and the peptide was synthesized by coupling amino acids one by one from the C-terminus to the N-terminus. The method is as follows:

[0049] (1) Place 2-Chlorotrityl Chloride Resin (i.e. 2-chlorotrityl chloride resin) into a reaction tube, add DMF (i.e. N,N-dimethylformamide) (15 ml / g), and shake for 60 min.

[0050] (2) Coupling the first amino acid at the C-terminus: Filter out the solvent through a sand core, add a 3-fold molar excess of Fmoc-protected amino acid (the first amino acid at the C-terminus), then add a 10-fold molar excess of DIEA (N,N-diisopropylethylamine), and finally add DMF to dissolve, shake for 30 minutes, cap with methanol, and react for 30 minutes.

[0051] (3) Deprotection: remove DMF, add 20% piperidine DMF solution (15 ml / g), react for 5 min, remove the solvent, add 20% piperidine DMF solution (15 ml / g), and react for 15 min.

[0052] (4) Detection: Drain the piperidine solution and take a dozen resin pellets for detection using the ninhydrin detection method. A dark blue color indicates a positive reaction.

[0053] (5) Washing: Take the resin coupled with the first amino acid, wash twice with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g).

[0054] (6) Condensation: Take the washed product and add a 3-fold molar excess of the second amino acid protected by Fmoc, a 3-fold molar excess of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), and then add a 10-fold molar excess of DIEA. Finally, add DMF to dissolve and shake for 45 min.

[0055] (7) Detection: Take a dozen resin pellets and test them using the ninhydrin test method. A colorless reaction indicates a negative reaction.

[0056] (8) Washing: Take the resin coupled with the first and second amino acids, wash once with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g).

[0057] (9) Continue coupling: According to the conventional steps of peptide solid phase synthesis and the conditions and methods of steps (3) to (8), carry out amino acid coupling and deprotection in sequence from the C-terminus to the N-terminus until the last amino acid coupling and Fmoc protecting group removal are completed. Wash the product: DMF (10 ml / g) twice, DCM (10 ml / g) three times, methanol (10 ml / g) four times, and drain for 10 min.

[0058] (10) Resin cleavage and cracking: Prepare cleavage solution (10 mL / g): TFA (i.e., trifluoroacetic acid) 95%, water 2%, EDT (i.e., ethanedithiol) 2%, and TIS (i.e., triisopropylsilane) 1%. The cleavage time is 180 min.

[0059] (11) Blow-drying and washing: The cleavage solution obtained in (10) was blown dry with nitrogen, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed with ether six times, and then evaporated at room temperature to obtain the crude peptide fragment.

[0060] (12) Purification and structure confirmation: The crude peptide was dissolved in H2O / CAN solution (CAN was fully dissolved in water at room temperature until it could no longer be dissolved; CAN is cerium ammonium nitrate). HPLC analysis was performed to determine the peak time corresponding to the target peak. HPLC analysis conditions:

[0061] The C18 reverse phase chromatography system was used, with a wavelength of 220 nm, a mobile phase flow rate of 15 ml / min, an injection volume of 20 μL, and a column temperature of 25 °C. Mobile phase A was 0.1% TFA aqueous solution; mobile phase B was 0.1% TFA acetonitrile solution. Results are shown in Figure 5 The retention time of the target peptide is 9.328min, and the purity is 99.34% calculated by peak area. Take a small amount of the target peak solution in a 1.5ml centrifuge tube for MS molecular weight identification and HPLC analysis purity identification. Its [M+H] + The mass-to-nuclear ratio (m / z) is 739.4Da.

[0062] Example 4 Solid Phase Synthesis of Soybean Meal Active Decapeptide AIPVNKPGRF

[0063] In this example, the oligopeptide (AIPVNKPGRF) with peptide number 2 was prepared by solid phase synthesis, and the peptide was synthesized by coupling amino acids one by one from the C-terminus to the N-terminus. The method is as follows:

[0064] (1) Place 2-Chlorotrityl Chloride Resin in a reaction tube, add DMF (15 ml / g), and shake for 60 min.

[0065] (2) Coupling the first amino acid at the C-terminus: Filter out the solvent through a sand core, add a 3-fold molar excess of Fmoc-protected amino acid (the first amino acid at the C-terminus), then add a 10-fold molar excess of DIEA (N,N-diisopropylethylamine), and finally add DMF to dissolve, shake for 30 minutes, cap with methanol, and react for 30 minutes.

[0066] (3) Deprotection: remove DMF, add 20% piperidine DMF solution (15 ml / g), react for 5 min, remove the solvent, add 20% piperidine DMF solution (15 ml / g), and react for 15 min.

[0067] (4) Detection: Drain the piperidine solution and take a dozen resin pellets for detection using the ninhydrin detection method. A dark blue color indicates a positive reaction.

[0068] (5) Washing: Take the resin coupled with the first amino acid, wash twice with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g).

[0069] (6) Condensation: Take the washed product and add a 3-fold molar excess of the second amino acid protected by Fmoc, a 3-fold molar excess of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), and then add a 10-fold molar excess of DIEA. Finally, add DMF to dissolve and shake for 45 min.

[0070] (7) Detection: Take a dozen resin pellets and test them using the ninhydrin test method. A colorless reaction indicates a negative reaction.

[0071] (8) Washing: Take the resin coupled with the first and second amino acids, wash once with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g).

[0072] (9) Continue coupling: According to the conventional steps of peptide solid phase synthesis and the conditions and methods of steps (3) to (8), carry out amino acid coupling and deprotection in sequence from the C-terminus to the N-terminus until the last amino acid coupling and Fmoc protecting group removal are completed. Wash the product: DMF (10 ml / g) twice, DCM (10 ml / g) three times, methanol (10 ml / g) four times, and drain for 10 min.

[0073] (10) Resin cleavage and cracking: Prepare cleavage solution (10 mL / g): TFA (i.e., trifluoroacetic acid) 95%; water 2%; EDT (i.e., ethanedithiol) 2%; TIS (i.e., triisopropylsilane) 1%. The cleavage time is 180 min.

[0074] (11) Blow-drying and washing: The cleavage solution obtained in (10) was blown dry with nitrogen, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed with ether six times, and then evaporated at room temperature to obtain the crude peptide fragment.

[0075] (12) Purification and structure confirmation: The crude peptide was dissolved in H2O / CAN solution (CAN was fully dissolved in water at room temperature until it could no longer be dissolved; CAN is cerium ammonium nitrate). HPLC analysis was performed to determine the peak time corresponding to the target peak. HPLC analysis conditions:

[0076] The C18 reverse phase chromatography system was used, with a wavelength of 220 nm, a mobile phase flow rate of 15 ml / min, an injection volume of 20 μL, and a column temperature of 25 °C. Mobile phase A was 0.1% TFA aqueous solution; mobile phase B was 0.1% TFA acetonitrile solution. Results are shown in Figure 6 The retention time of the target peptide was 6.363 min, and the purity was 98.77% calculated based on the peak area.

[0077] Use a 1.5 ml centrifuge tube to take a small amount of the target peak solution for MS molecular weight identification and HPLC analysis purity identification. + The mass-to-nuclear ratio (m / z) is 1098.6Da.

[0078] Example 5 DPP-IV Inhibitory Effect of Soybean Meal Peptides

[0079] Experimental samples and preparation:

[0080] The peptide powder mixture prepared in Example 1 was diluted with pure water to prepare a 5 mg / mL initial solution as a soybean meal crude peptide control. The KLGKFF and AIPVNKPGRF oligopeptides prepared in Examples 3 and 4 were diluted with pure water to prepare 5 mg / mL initial solutions as two experimental samples. The DPP-IV inhibitor sitagliptin was used as a positive control, and the sitagliptin concentration was 1.25 mg / L (pure water dilution). The DPP-IV enzyme inhibition activity of each group was measured.

[0081] The DPP-IV enzyme inhibition of the test substance was determined according to the method and formula in the literature (Yu, Yang, et al. "Characterization and evaluation of the in vitro and in vivo anti-diabetic activities of camel milk protein hydrolysates derived with different proteasedigestions." Journal of Functional Foods 117 (2024): 106227.). The substrate for DPP-IV enzyme activity determination was 1.5mmol / L Gly-Pro-p-nitroanilide solution (prepared with pH=8.0, 100mmol / L Tris-HCL buffer), and the blank group did not add sitagliptin, soybean meal crude peptide or two oligopeptides.

[0082] The volume of each test substance added to the ELISA plate was 15 μL, the volume of DPP-IV enzyme (2U) added was 5 μL, the volume of 20 mmol / L Tris-HCl buffer at pH 8.0 was 50 μL (70 μL for the blank group and 65 μL for the positive control group), and the volume of substrate added was 30 μL. After incubation at 37°C, the fluorescence intensity of the reaction release product was measured at a wavelength of 380 nm using an ELISA reader. Three replicate wells were set up for each group to calculate the DPP-IV enzyme inhibition.

[0083] The inhibition rates of soybean meal crude peptide, KLGKFF, AIPVNKPGRF and sitagliptin on DPP-IV were significantly higher than those of the blank group, which were 44.30±1.50%, 77.19±2.07%, 82.88±0.93% and 89.28±0.79%, respectively. The inhibitory effect of KLGKFF and AIPVNKPGRF on DPP-IV enzyme was significantly higher than that of soybean meal crude peptide (p<0.05), and there was no significant difference in the inhibitory effect of KLGKFF and AIPVNKPGRF on DPP-IV enzyme.

[0084] Example 6 Effects of soybean meal peptides on the viability and glucose consumption of insulin-resistant HePG2 cells

[0085] 1. Establishment of insulin resistance (IR) HepG2 cell model:

[0086] This section is used to explore the conditions for establishing an insulin-resistant HepG2 cell model. The determination of glucose consumption in HepG2 cells refers to literature such as "Effects of Sweet Corn Cob Polysaccharides on Glucose Metabolism Function of Insulin-Resistant HepG2 Cells" (Ma Yongqiang et al. "Food Science" Issue 17, 2021).

[0087] HepG2 cells were taken out of the -80℃ freezer and revived routinely. The culture medium was DMEM high-glucose culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. When the cell density covered 80%~90% of the bottom of the culture flask, they were digested with 0.25% EDTA trypsin and passaged at a ratio of 1:3.

[0088] In order to determine the optimal dose of insulin and duration of treatment, when the cells are in the logarithmic growth phase, the culture medium is replaced with culture medium containing different concentrations of insulin to avoid excessive concentrations that affect cell viability, resulting in a decrease in cell number and a decrease in cell glucose consumption. -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 mol / L, MTT colorimetric method was used to verify the effect of insulin on the viability of HepG2 cells. -5 ~10 -8 M had no toxicity to HepG2 cells ( Figure 7 ). This concentration range was selected to culture the cells for 24 h. After the culture, glucose consumption was measured using a glucose kit to evaluate the effectiveness of insulin in promoting glucose uptake in HepG2 cells. - 5 mol / L insulin-treated cells for 24 h showed the highest glucose consumption rate ( Figure 8 ), indicating successful induction of insulin resistance. Therefore, this concentration was selected to establish the insulin resistance model, namely the IR model.

[0089] 2. Effects of soybean meal peptides on cell viability and glucose consumption in IR HepG2 cell model

[0090] 2.1 Effect of soybean meal peptide on HepG2 cell viability (MTT colorimetric method)

[0091] HepG2 cells in the logarithmic growth phase were taken, the culture medium was discarded, washed with PBS, and digested with 0.25% trypsin to prepare a cell suspension with a density of about 1×10 5 / mL. The cell suspension was inoculated in a 96-well plate, 150 μL was added to each well, 6 replicate wells were set up, and cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours. After the cells adhered to the wall, 150 μL of complete culture medium was added to the blank group; 150 μL of component a (the peptide powder mixture prepared in Example 1, i.e., crude soybean meal peptide) or KLGKFF or AIPVNKPGRF peptide solution of different concentrations (12.5, 25, 50, 100, 200, 400 μg / mL, dissolved in complete culture medium) was added to the sample group, and culture was continued for 24 hours. After the culture, the cell viability was determined by the conventional MTT method. The experimental results are shown in the following figure. Fig. 9 As shown in the figure, the cell viability of the samples after treatment was above 90%, and the cell viability of the KLGKFF group was higher than that of the AIPVNKPGRF group; soybean meal crude peptides and active peptides had no significant inhibitory effect on the viability of HepG2 cells, and had good safety.

[0092] 2.2 Effect of soybean meal peptide on glucose consumption in IR HepG2 cell model

[0093] This part is used to investigate the effect of soybean meal peptides on glucose consumption in insulin-resistant HepG2 cell models. The determination of cellular glucose consumption was based on literature such as "Effects of Sweet Corn Cob Polysaccharides on Glucose Metabolism Function of Insulin-Resistant HepG2 Cells" (Ma Yongqiang et al., "Food Science", Issue 17, 2021).

[0094] The IR HepG2 cell model was established according to the method in Part 1 of this example.

[0095] The experiment was divided into a blank group (i.e., normal HepG2 cells), an IR model group (IR HepG2 cells), and a group consisting of KLGKFF peptide, AIPVNKPGRF peptide, and group a (peptide powder mixture prepared in Example 1, i.e., crude soybean meal peptide). Referring to the literature method (Ma Yongqiang et al. Effect of sweet corn cob polysaccharide on glucose metabolism function of insulin-resistant HepG2 cells. Food Science, Issue 17, 2021), normal HepG2 cells and IR HepG2 cells were prepared into cell suspensions with a density of about 1x10 5 / mL, the cell suspension was inoculated in a 24-well plate, 500μL was added to each well, and 3 replicates were set up. The cells were cultured in a cell culture incubator at 37℃ and 5% CO2 for 12h. After the cells adhered to the wall, they were first treated with insulin modeling and then intervened with soybean meal peptide for 24h. The blank group was added with cell-free culture medium (high-glucose DMEM containing 10% FBS and 1% double antibody); the model group: cells induced by insulin but not treated with soybean meal peptide, added with cell-free culture medium; the sample group: cells induced by insulin and treated with different concentrations of sample peptide (12.5~400μg / L). Each group was set up with 3 replicates. After incubation in a 37℃, 5% CO2 incubator for 48h, the glucose content in the supernatant of the culture medium (high-glucose DMEM containing 10% FBS and 1% double antibody) was detected with a glucose kit to calculate the glucose consumption.

[0096] Under IR conditions, the glucose consumption of cells will be weakened, which will increase the blood glucose concentration and develop into type 2 diabetes. Therefore, the glucose consumption of the IR HepG2 cell model can be used to investigate the intervention effect of drugs on type 2 diabetes. Fig.10 As shown in the figure, compared with the blank group, the glucose consumption in the model group was significantly reduced, indicating that a stable IR HepG2 model was successfully established. Compared with the model group, the administration of soybean meal peptides significantly increased the glucose consumption of cells (compared with the model group, p < 0.05). Among them, the glucose consumption in the AIPVNKPGRF and KLGKFF groups was significantly higher than that in the a component (i.e., soybean meal crude peptides).

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, the above-mentioned embodiments can also be modified, combined and improved without departing from the concept of the present invention, and these all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims

1. Use of an oligopeptide having DPP-IV inhibitory activity in the preparation of a hypoglycemic active agent, characterized in that: The amino acid sequence of the oligopeptide is shown in SEQ ID NO.

1.

2. The use of an oligopeptide having DPP-IV inhibitory activity according to claim 1 in the preparation of a hypoglycemic active agent, characterized in that: The hypoglycemic active agent is any one of an inhalation preparation, an external preparation, an injection preparation, an implant, and an oral preparation.

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