Camel milk active peptides and their use in preparing drugs for treating and / or preventing diabetes

Abstract

The present invention provides camel milk active peptides and their use in preparing a drug for treating and / or preventing diabetes. The present invention obtains two polypeptide compounds from the papain hydrolysis product of fresh camel milk. Biological activity evaluation revealed that these polypeptide compounds have significant protein tyrosine phospholipase 1B (PTP1B) inhibitory activity, can enhance insulin receptor sensitivity, and promote glucose uptake in hepatocytes. These compounds have druggable potential for treating type 2 diabetes, providing a new material basis for developing innovative drugs for treating type 2 diabetes and innovative hypoglycemic drugs such as PTP1B inhibitors.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a camel milk active peptide and application thereof in preparing a drug for treating and / or preventing diabetes. Background Art

[0002] Diabetes is a chronic endocrine and metabolic disease, in which PTP-1B is a key negative regulator in the insulin signaling pathway. PTP-1B inhibitors block insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR), thereby affecting the phosphorylation of the insulin receptor (IRS-1), sensitizing insulin-like and insulin-like substances and lowering blood sugar. Simultaneously, they can enhance leptin signaling, inducing increased fat metabolism and weight loss.

[0003] Insulin resistance is a key factor in the development of type 2 diabetes, and protein tyrosine phosphatase 1B (PTP1B) plays a crucial role in the development of insulin resistance. Studies have shown that PTP1B, as a negative regulator of the insulin signaling pathway, can dephosphorylate tyrosine residues of activated insulin receptors or their substrates, thereby terminating insulin signaling and causing insulin resistance, leading to relative insulin deficiency and the development of type 2 diabetes. Animal studies have shown that PTP1B knockout mice exhibit significantly improved insulin sensitivity in glucose tolerance and insulin tolerance tests. Therefore, PTP1B inhibitors may play an important role as a novel therapeutic agent for type 2 diabetes.

[0004] Therefore, it is of great significance to develop innovative hypoglycemic drugs such as PTP1B inhibitors and their drugs for the treatment and prevention of diabetes. Summary of the Invention

[0005] In view of this, the present invention provides a camel milk active peptide and its use in preparing a drug for treating and / or preventing diabetes.

[0006] The technical solution of the present invention is achieved as follows:

[0007] Application of camel milk active peptides in the preparation of drugs for treating and / or preventing diabetes.

[0008] Application of camel milk active peptides in the preparation of PTP1B inhibitors.

[0009] Furthermore, the camel milk active peptide includes compound I with an amino acid sequence of LPLLLPR and / or compound II with an amino acid sequence of ELILDIK.

[0010] Furthermore, the structure of the compound I is shown in Formula I:

[0011]

[0012] Furthermore, the structure of compound II is shown in Formula II:

[0013]

[0014] Furthermore, the drug includes at least one of Compound I and its pharmaceutically acceptable salt and / or Compound II and its pharmaceutically acceptable salt.

[0015] Furthermore, the drug includes one or more pharmaceutically acceptable excipients.

[0016] Furthermore, the drug is selected from one of injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The active peptides LPLLLPR and ELILDIK in camel milk exhibit inhibitory activity against PTP1B at relatively low concentrations, exhibiting anti-diabetic activity and potential as drug candidates for the treatment of type 2 diabetes. This provides a novel material foundation for the development of innovative drugs for the treatment of type 2 diabetes and innovative hypoglycemic drugs such as PTP1B inhibitors. Furthermore, this invention provides candidate compounds for the research and development of new PTP1B inhibitors and drugs for the treatment of type 2 diabetes, and provides a scientific basis for the development and utilization of enzymatically hydrolyzed natural active substances derived from camel milk. The two active peptides, LPLLLPR and ELILDIK, are isolated from widely consumed camel milk, and their safety is preliminarily assured. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the high performance liquid chromatogram of LPLLLPR of the present invention.

[0020] Figure 2 It is the high performance liquid chromatography-mass spectrum of LPLLLPR of the present invention.

[0021] Figure 3 It is the high performance liquid chromatogram of ELILDIK of the present invention.

[0022] Figure 4 is the high performance liquid chromatography-mass spectrometry diagram of ELILDIK of the present invention.

[0023] Figure 5 is the LPLLLPR( Figure 5 A)LMPSGIL( Figure 5 B),AVPYPQR( Figure 5 C) ELILDIK Figure 5 D), WIEEEAVR( Figure 5 E) and the positive drug Na3VO4( Figure 5F) Inhibitory activity test results against PTP1B.

[0024] Figure 6 The different concentrations of LPLLLPR ( Figure 6 A) and ELILDIK( Figure 6 B) Effects on HepG2 cell viability (n=4, ).

[0025] Figure 7 The effects of different concentrations of LPLLLPR and ELILDIK on glucose consumption in IR HepG2 cell models (n=4, ).

[0026] Note: Compared with the Control group: #P<0.05, ##P<0.01; Compared with the INS group: *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0030] The papain of the present invention is produced by Nanning Pangbo Bioengineering Co., Ltd. and its product name is papain.

[0031] Example 1 Preparation and Identification of Camel Milk Active Peptides

[0032] (1) Fresh camel milk from Xinjiang was centrifuged at 8000 rpm and 10°C for 30 min, and the milk was taken out. The upper fat layer and the lower sediment were removed to separate the total protein of the camel milk. The total protein of the camel milk was stored in a refrigerator at -20°C for later use.

[0033] (2) Add 8000U / g papain to the camel milk total protein and perform enzymolysis at a temperature of 65°C and a pH of 7.0 for 3h. After the enzymolysis is completed, the temperature of the enzymolysis solution is raised to 95°C and maintained for 10min to inactivate the enzyme to stop the reaction. The enzymolysis solution is quickly cooled to room temperature and then adjusted to pH 7.0 with 2mol / L dilute hydrochloric acid or 2mol / L sodium hydroxide solution. The solution is centrifuged at 5000rpm and 4°C for 5min and the supernatant is collected.

[0034] (3) ultrafiltration of the supernatant to collect polypeptide solutions less than 10 kDa, rotary evaporation concentration, and freeze-drying to obtain polypeptide freeze-dried powder;

[0035] (4) The polypeptide freeze-dried powder obtained in step (3) was separated and purified by a Superdex™ peptide 10 / 300GL gel column; the mobile phase for separation was 0.1% (volume fraction) trifluoroacetic acid in 30% (volume fraction) acetonitrile, the flow rate was 1 mL / min, the protein chromatography wavelength was 280 nm, the elution peak was collected, and freeze-dried to obtain the separated polypeptide;

[0036] (5) The separated polypeptide obtained in step (4) was separated again by a reversed-phase high-performance liquid column; the liquid A used in the liquid phase was: 0.1% (volume fraction) formic acid aqueous solution, the liquid B was: 0.1% (volume fraction) formic acid acetonitrile aqueous solution (acetonitrile volume fraction was 84%), and the chromatographic column was RP-HPLC C18 (150 mm × 0.15 mm, 5 μm); the column temperature was room temperature, the flow rate was 0.25 mL / min; the mobile phase was a gradient: 0-50 min (liquid B; 4%-50% volume fraction), 50-54 min (liquid B; 50%-80% volume fraction), 54-60 min (liquid B; 100% volume fraction), the ultraviolet detection wavelength was 214 nm, and the components were collected according to the peaks to obtain camel milk polypeptide 1 and camel milk polypeptide 2;

[0037] (6) Structural identification of the camel milk polypeptide (<10 KD) obtained from the polypeptide freeze-dried powder of step (3) and the camel milk polypeptide 1 and camel milk polypeptide 2 obtained in step (5) was performed using UPLC-ESI-MS / MS; Ultra-high performance liquid chromatography (UPLC) conditions: chromatographic column Popular PS-C18 (250×0.075 mm, 3 μm, pore size 100 Å); mobile phase A was an aqueous solution containing 0.1% (volume fraction) formic acid and 2% (volume fraction) acetonitrile; mobile phase B was an acetonitrile solution containing 0.1% (volume fraction) formic acid and 10% (volume fraction) water; mass spectrometry conditions: the mass spectrometry ion source voltage was set to 2.0 kV, the primary mass spectrometry scan range was set to 400-1800 m / z, and the scan resolution was set to 70,000; the secondary mass spectrometry scan resolution was set to 35,000; the data acquisition mode used the data-dependent scan (DDA) program, and the top 10 peptide precursors with the highest signal intensity were selected after the primary scan. The ions were sequentially introduced into the HCD collision cell for fragmentation using 30% fragmentation energy. To improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 1E5, the signal threshold was set to 20,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of the parent ion. The LC / MS results were analyzed with the help of MaxQuant software. By comparing with the Bactrian camel protein sequences in the Uniprot protein database, 44 camel milk polypeptides (<10 KD) were identified, as well as camel milk polypeptide 1 (LPLLLPR) and camel milk polypeptide 2 (ELILDIK).

[0038] (7) Using Discovery studio2019 molecular docking software, molecular docking was performed with 44 peptides as ligands and PTP1B protein (PDB:) as receptor. According to the docking scores, the peptides with the best PTP1B binding activity were selected: LMPSGIL, AVPYPQR, WIEEEAVR, LPLLLPR and ELILDIK.

[0039] Among them, after chromatographic analysis, the peak table information corresponding to the high performance liquid chromatogram of camel milk active peptide LPLLLPR is shown in Table 1 below:

[0040] Table 1 Peak information of camel milk active peptide LPLLLPR

[0041]

[0042] Its high performance liquid chromatogram is as follows Figure 1 As shown, the mass spectrum (MW: 821.07, m / z: 821.50+1, 411.50+2) is as follows Figure 2As shown, after analysis, it can be concluded that the compound structure of camel milk active peptide LPLLLPR is:

[0043]

[0044] After chromatographic analysis, the peak table information corresponding to the HPLC chromatogram of camel milk active peptide ELILDIK is shown in Table 2 below:

[0045] Table 2 Peak information of camel milk active peptide ELILDIK

[0046]

[0047] The HPLC chromatogram of camel milk active peptide ELILDIK is as follows: Figure 3 As shown, the mass spectrum (MW: 843.03, m / z: 843.40+1, 422.35+2) is as follows Figure 4 As shown, after analysis, it can be concluded that the compound structure of camel milk active peptide ELILDIK is:

[0048]

[0049] Example 2 Protein tyrosine phospholipase 1B (PTP1B) inhibitory activity assay

[0050] Camel milk polypeptides 1 (LPLLLPR) and 2 (ELILDIK), as well as peptides LMPSGIL, AVPYPQR, and WIEEEAVR, were used as samples to determine their inhibitory activity against protein tyrosine phospholipase 1B (PTP1B). Disodium p-nitrophenyl phosphate (p-NPP) was used as a substrate, and sodium orthovanadate (Na3VO4) was used as a positive control. A microplate reader was used to screen PTP1B inhibitors. The activity of PTP1B was determined by the color reaction produced by the hydrolysis of the phosphate group of p-NPP by PTP1B.

[0051] LPLLLPR, ELILDIK, LMPSGIL, AVPYPQR, WIEEEAVR and sodium orthovanadate (Na3VO4) were prepared as follows Figure 5 Sample solutions of different concentrations were prepared. 20 μL of sample solutions and PTP1B enzyme solution of each concentration were added to a 96-well plate. After warming to 37°C for 5 minutes, 160 μL of buffer solution containing substrate (33 mmol / L) was added. After incubation at 37°C for 30 minutes, 2 M NaOH solution was added to terminate the reaction. Three replicate wells were set for each group. The inhibition rate was calculated according to the following formula:

[0052] Inhibition rate % = [(A blank - A inhibition) / A blank] × 100%

[0053] in:

[0054] A Inhibition: Absorbance measured by PTP1B enzyme solution + p-NPP + sample solution + NaOH solution

[0055] A blank: absorbance measured by PTP1B enzyme solution + p-NPP + distilled water + NaOH solution

[0056] The results of the test are as follows Figure 5 As shown, according to Figure 5 It can be seen that the peptides LMPSGIL and AVPYPQR had almost no inhibitory effect on PTP1B, and the peptide WIEEEAVR did not achieve a 50% inhibitory effect on PTP1B at a concentration of 10 mg / mL; while the IC values of LPLLLPR and ELILDIK for PTP1B inhibition were 50 The values were 14.33 mg / mL and 4.85 mg / mL, respectively. LPLLLPR and ELILDIK can exert their inhibitory effects on PTP1B at lower concentrations, have anti-diabetic activity, and have drug-forming potential in the treatment of type 2 diabetes. This provides a new material basis for the development of innovative drugs for the treatment of type 2 diabetes and the development of innovative hypoglycemic drugs such as PTP1B inhibitors.

[0057] Example 3 Experiment on Glucose Uptake by Hepatocytes by LPLLLPR and ELILDIK

[0058] (1) Culture of HepG2 cells

[0059] Cell recovery: After taking out the frozen HepG2 cells from the -150℃ freezer, immediately place them in a 37℃ constant temperature water bath to thaw, and shake the cryovial constantly during the process to allow it to thaw quickly and evenly; after the cell suspension in the cryovial is thawed, take the cryovial to the clean bench, gently open the cryovial cap, use a 1mL pipette to transfer the cell suspension in the cryovial to a 15mL centrifuge tube, and add 5 times the volume of DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture) at the same time, and centrifuge at 1000rpm for 5min; after centrifugation, discard the supernatant, add 1mL DMEM complete medium and gently pipette to mix; transfer 1mL of cell suspension to a T25 culture flask containing 5mL DMEM complete medium and shake slowly to ensure uniform distribution of cells; finally, place the culture flask in a 37℃, 5% CO2 incubator for culture, and replace the medium after the cells attach to the wall;

[0060] Passaging: Remove the culture flask from the incubator and observe the cell growth under a microscope. When the cell coverage in the culture flask reaches more than 80%, discard the culture medium and wash once with 1× PBS buffer; then add about 1 mL of trypsin (containing EDTA) to the culture flask to digest the cells and observe the cell morphology under an optical microscope. When the cells become round and the volume decreases, immediately add 2-3 mL of DMEM complete medium to terminate the digestion; finally, use a 1 mL pipette to pipette the cells to the wall surface to suspend all the adherent cells into a cell suspension; place the cell suspension in a centrifuge tube and centrifuge at 1000 rpm for 5 minutes; after centrifugation, discard the supernatant, add 2 mL of DMEM complete medium and gently pipette to mix, then divide the cell suspension into two and inoculate them into new culture flasks to complete the passaging;

[0061] Cryopreservation: After digesting the cells as described above, place the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the culture medium and resuspend the cells in cell freezing solution (50% complete culture medium + 40% FBS + 10% DMSO), then aliquot into cryopreservation tubes. After the cryopreservation tubes have been allowed to stand for a few minutes, place them in a gradient cooling box and place them in a -150°C refrigerator for one day before transferring them to a cryopreservation box for long-term storage.

[0062] (2) Effects of two peptides on HepG2 cell viability

[0063] Cells were plated at 1×10 4 The cells were seeded into a 96-well plate at a concentration of 100 μg / mL and pre-incubated overnight. After pre-incubation, HepG2 cells were exposed or not exposed to drugs for 24 hours (1.25 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, and 40.0 μg / mL of PTP1B inhibitory peptide). The culture medium was discarded, and 100 μL of 10% CCK-8 solution was added to each well. The cells were incubated in a 37°C oven for 1-2 hours until the liquid turned orange-yellow. The absorbance value was detected at a wavelength of 450 nm using a microplate reader. The results are shown in Figure 2. Figure 6 shown.

[0064] The results show that Figure 6 In A, compared with the blank group, LPLLLPR had no cytotoxicity in the concentration range of 1.25 to 40 μg / mL, and at the concentrations of 2.5 and 5 μg / mL, LPLLLPR significantly promoted the proliferation of HepG2 cells after 24 h of culture (P<0.05); Figure 6In B, compared with the blank group, ELILDIK had no cytotoxicity in the concentration range of 1.25 to 40 μg / mL, and when the concentration of ELILDIK was 5 and 10 μg / mL, it could significantly promote the proliferation of HepG2 cells after 24 hours of culture (P<0.05).

[0065] (3) Effects of two peptides on glucose uptake in IR HepG2 cells

[0066] The effects of LPLLLPR and ELILDIK on glucose consumption in the IR HepG2 cell model were investigated.

[0067] Cells were plated at 1×10 4 The cells were seeded into 96-well plates and pre-incubated overnight. After pre-incubation, HepG2 cells were exposed to 10 -6 M insulin, cells not treated with insulin served as the control group. After 36 hours of incubation, cells were exposed or not exposed to the drug and incubated for 24 hours to detect the glucose content in the supernatant. The highest glucose concentration in each group of cells was 22.5 mmol / L. The glucose content in the final supernatant was measured by the glucose oxidase-peroxidase coupled method (GOD-POD). The specific operation method was referred to the instructions of the glucose kit. The results are shown in the figure. Figure 7 Glucose consumption is calculated as follows:

[0068] Glucose consumption = glucose content of blank culture medium - glucose content of experimental group

[0069] Figure 7 The results showed that compared with the blank group, glucose consumption in the model group was significantly reduced (P < 0.05), indicating that the IRHepG2 cell model was successfully established. Compared with the model group, different concentrations of LPLLLPR and ELILDIK significantly increased glucose consumption in IRHepG2 cells (P < 0.05), and glucose consumption increased with increasing concentration. This suggests that LPLLLPR and ELILDIK can effectively improve glucose-induced insulin resistance in HepG2 cells.

[0070] In summary, the LPLLLPR and ELILDIK obtained in the present invention effectively inhibit PTP1B and have certain blood sugar lowering potential, and can have drug development potential in the treatment of type 2 diabetes, providing a new material basis for the development of innovative drugs for the treatment of type 2 diabetes and the development of innovative hypoglycemic drugs such as PTP1B inhibitors.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of camel milk active peptides in the preparation of drugs for treating diabetes, wherein the camel milk active peptides are selected from one or both of compound I with the amino acid sequence LPLLLPR and compound II with the amino acid sequence ELILDIK.

2. The use according to claim 1, characterized in that The structure of the compound I is shown in Formula I: 。 3. The use according to claim 1, characterized in that The structure of compound II is shown in formula II: 。 4. The use according to claim 1, characterized in that The drug comprises at least one of compound I and its pharmaceutically acceptable salt and / or compound II and its pharmaceutically acceptable salt.

5. The use according to claim 1, characterized in that The drug includes one or more pharmaceutically acceptable excipients.

6. The use according to claim 1, characterized in that The medicine is selected from one of injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

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