Camel milk active peptide and application thereof in preparation of drugs and edible products for treating and / or preventing diabetes

Abstract

The invention provides camel milk active peptide and application thereof in preparation of drugs and edible products for treating and / or preventing diabetes mellitus. Two polypeptide compounds are obtained from papain enzymolysis products of fresh camel milk. Bioactivity evaluation shows that the polypeptide compound has remarkable protein tyrosine phospholipase 1B (PTP1B) inhibitory activity, can enhance insulin receptor sensitivity and promote hepatocyte glucose uptake, has patent medicine potential in the aspect of treating type II diabetes, and can be used for preparing medicines for treating type II diabetes. And a new material basis is provided for developing innovative medicines for treating type II diabetes and developing innovative PTP1B inhibitor hypoglycemic medicines.

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 medicines and edible products for treating and / or preventing diabetes. Background Art

[0002] Diabetes is a chronic endocrine metabolic disease, in which PTP-1B is a key negative regulator protein in the insulin signal transduction pathway. PTP-1B inhibitors block the tyrosine phosphorylation of insulin receptor (IR) stimulated by insulin, thereby affecting the phosphorylation of insulin receptor (IRS-1), sensitizing insulin-like and insulin, and lowering blood sugar. At the same time, it can enhance leptin signals, induce increased fat metabolism levels, and reduce body weight.

[0003] Insulin resistance is a key factor in the development of type 2 diabetes, and protein tyrosine phosphatase 1B (PTP1B) plays a key role in the formation of insulin resistance. Studies have shown that PTP1B, as a negative regulator of the insulin signaling pathway, can dephosphorylate the tyrosine residues of activated insulin receptors or their substrates, thereby terminating the insulin signaling pathway, causing the body to resist insulin, resulting in a relative lack of insulin in the body and then developing type 2 diabetes. Animal studies have shown that PTP1B knockout mice show a significant increase in insulin sensitivity in glucose tolerance and insulin tolerance tests. Therefore, PTP1B inhibitors can play an important role as a new type of drug for the treatment of 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 application in preparing a medicine and edible product for treating and / or preventing diabetes.

[0006] The technical solution of the present invention is achieved in this way:

[0007] Application of camel milk active peptides in the preparation of edible products.

[0008] Application of camel milk active peptides in the preparation of drugs for treating and / or preventing diabetes.

[0009] Application of camel milk active peptides in the preparation of PTP1B inhibitors.

[0010] Furthermore, the camel milk active peptides include compound I with an amino acid sequence of LPLLLPR and / or compound II with an amino acid sequence of ELILDIK.

[0011] Furthermore, the structure of the compound I is as shown in Formula I:

[0012]

[0013] Furthermore, the structure of the compound II is as shown in Formula II:

[0014]

[0015] Furthermore, the drug includes at least one of Compound I and its pharmaceutically acceptable salt and / or Compound II and its pharmaceutically acceptable salt.

[0016] Furthermore, the edible product is selected from one of oral liquid, tea, lozenge, capsule, drink or effervescent tablet.

[0017] Furthermore, the drug includes one or more pharmaceutically acceptable excipients.

[0018] Furthermore, the drug is selected from one of injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The active LPLLLPR and ELILDIK in camel milk show inhibitory activity on PTP1B at lower concentrations, have anti-diabetic activity, and have drug-making 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. In addition, the present 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 enzymatic natural active substances derived from camel milk. The above two active peptides LPLLLPR and ELILDIK are separated from camel milk, which is widely consumed by the public, and their safety is preliminarily guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the high performance liquid chromatogram of LPLLLPR of the present invention.

[0022] Figure 2 It is the high performance liquid chromatography-mass spectrum of LPLLLPR of the present invention.

[0023] Figure 3 It is the high performance liquid chromatogram of ELILDIK of the present invention.

[0024] Figure 4 It is the high performance liquid phase mass spectrum of ELILDIK of the present invention.

[0025] Figure 5 is the LPLLLPR( Figure 5A)LMPSGIL( Figure 5 B)AVPYPQR( Figure 5 C) ELILDIK Figure 5 D) WIEEEAVR ( Figure 5 E) and positive drug Na3VO4( Figure 5 F) The results of the inhibitory activity test on PTP1B.

[0026] Figure 6 The different concentrations of LPLLLPR ( Figure 6 A) and ELILDIK( Figure 6 B) Effects on the viability of HepG2 cells (n=4, x±s).

[0027] Figure 7 This is the effect of different concentrations of LPLLLPR and ELILDIK of the present invention on the glucose consumption of IRHepG2 cell model (n=4, x±s).

[0028] 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

[0029] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0030] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0031] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0032] The papain of the present invention is produced by Nanning Pangbo Bioengineering Co., Ltd. and its product name is papain.

[0033] Example 1 Preparation and identification of camel milk active peptides

[0034] (1) fresh camel milk from Xinjiang was centrifuged at 8000 rpm and 10° C. for 30 min, and the milk was taken out, and the upper fat layer and the lower sediment layer were removed to separate the total camel milk protein; the total camel milk protein was stored in a refrigerator at -20° C. for later use;

[0035] (2) adding 8000U / g papain to the camel milk total protein, performing enzymolysis at a temperature of 65°C and a pH of 7.0 for 3h, raising the temperature of the enzymolysis solution to 95°C and maintaining it for 10min to inactivate the enzyme to stop the reaction, rapidly cooling the enzymolysis solution to room temperature, adjusting the pH to 7.0 with 2mol / L dilute hydrochloric acid or 2mol / L sodium hydroxide solution, centrifuging at 5000rpm and 4°C for 5min, and taking the supernatant;

[0036] (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;

[0037] (4) separating and purifying the polypeptide freeze-dried powder obtained in step (3) by passing it through a SuperdexTMpeptide 10 / 300GL gel column; the mobile phase for separation is 0.1% (volume fraction) trifluoroacetic acid in 30% (volume fraction) acetonitrile, the flow rate is 1 mL / min, the detection wavelength of the protein chromatograph is 280 nm, the elution peak is collected, and freeze-dried to obtain the separated polypeptide;

[0038] (5) The separated polypeptide obtained in step (4) is separated again by a reversed-phase high-performance liquid column; the liquid A used in the liquid phase is: 0.1% (volume fraction) formic acid aqueous solution, the liquid B is: 0.1% (volume fraction) formic acid acetonitrile aqueous solution (acetonitrile volume fraction is 84%), and the chromatographic column is RP-HPLC C18 (150 mm×0.15 mm, 5 μm); the column temperature is room temperature, the flow rate is 0.25 mL / min; the mobile phase is 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 is 214 nm, and the components are collected according to the peaks to obtain camel milk polypeptide 1 and camel milk polypeptide 2;

[0039] (6) Using UPLC-ESI-MS / MS to identify the camel milk polypeptide (<10KD) obtained from the polypeptide freeze-dried powder of step (3), camel milk polypeptide 1 and camel milk polypeptide 2 obtained in step (5); ultra-high performance liquid chromatography (UPLC) conditions: chromatographic column PopularPS-C18 (250×0.075mm, 3μm, pore size 100A); mobile phase A is an aqueous solution containing 0.1% (volume fraction) formic acid and 2% (volume fraction) acetonitrile; mobile phase B is an acetonitrile solution containing 0.1% (volume fraction) formic acid and 10% (volume fraction) water; mass spectrometry conditions: mass spectrometry ion source voltage is set to 2.0kV, primary mass spectrometry scanning range is set to 400-1800m / z, scanning resolution is set to 70,000; secondary mass spectrometry scanning resolution is set to The data acquisition mode used the data-dependent scanning (DDA) program. After the first-level scan, the top 10 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn and fragmented using 30% fragmentation energy. In order 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 precursor 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 (<10KD) and camel milk polypeptide 1 (LPLLLPR) and camel milk polypeptide 2 (ELILDIK) were identified.

[0040] (7) Discovery studio2019 molecular docking software was used to perform molecular docking with 44 peptides as ligands and PTP1B protein (PDB:) as receptor. The peptides with the best PTP1B binding activity were selected based on the docking scores: LMPSGIL, AVPYPQR, WIEEEAVR, LPLLLPR and ELILDIK.

[0041] 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:

[0042] Table 1 Peak information of camel milk active peptide LPLLLPR

[0043]

[0044] 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 2 As shown, after analysis, it can be concluded that the compound structure of camel milk active peptide LPLLLPR is:

[0045]

[0046] After chromatographic analysis, the peak table information corresponding to the HPLC chromatogram of camel milk active peptide ELILDIK is shown in Table 2 below:

[0047] Table 2 Peak information of camel milk active peptide ELILDIK

[0048]

[0049] 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:

[0050]

[0051] Example 2 Protein tyrosine phospholipase 1B (PTP1B) inhibitory activity assay

[0052] Camel milk polypeptide 1 (LPLLLPR) and camel milk polypeptide 2 (ELILDIK), polypeptide LMPSGIL, polypeptide AVPYPQR, and polypeptide WIEEEAVR were used as samples to determine their inhibitory activity against protein tyrosine phospholipase 1B (PTP1B). During the determination, disodium p-nitrophenyl phosphate (p-NPP) was used as a substrate, sodium orthovanadate (Na3VO4) was used as a positive control, and an ELISA was used to screen PTP1B enzyme inhibitors. The activity of PTP1B was determined based on the color reaction produced by the hydrolysis of the phosphate group of p-NPP by PTP1B;

[0053] LPLLLPR, ELILDIK, LMPSGIL, AVPYPQR, WIEEEAVR and sodium orthovanadate (Na3VO4) were prepared as follows: Figure 5 Sample solutions of different concentrations were prepared, and 20 μL of sample solutions of each concentration and PTP1B enzyme solution were added to a 96-well plate. After warming to 37°C for 5 min, 160 μL of buffer solution containing substrate (33 mmol / L) was added. After incubation at 37°C for 30 min, 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:

[0054] Inhibition rate % = [(A blank - A inhibition) / A blank] × 100%

[0055] in:

[0056] A Inhibition: absorbance measured by PTP1B enzyme solution + p-NPP + sample solution + NaOH solution

[0057] A Blank: absorbance measured by PTP1B enzyme solution + p-NPP + distilled water + NaOH solution

[0058] 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 have almost no inhibitory effect on PTP1B, and the peptide WIEEEAVR does not reach a 50% inhibitory effect on PTP1B at a concentration of 10 mg / mL; while the IC values ​​of LPLLLPR and ELILDIK for PTP1B inhibition are 50 The values ​​were 14.33 mg / mL and 4.85 mg / mL respectively. LPLLLPR and ELILDIK can exert their inhibitory effect on PTP1B at lower concentrations, have anti-diabetic activity, and have drug-making 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.

[0059] Example 3 Experiment on glucose absorption of hepatocytes by LPLLLPR and ELILDIK

[0060] (1) Culture of HepG2 cells

[0061] Cell recovery: After taking out the frozen HepG2 cells from the -150℃ refrigerator, immediately place them in a 37℃ constant temperature water bath to thaw, and shake the cryotube continuously during the process to allow it to melt quickly and evenly; after the cell suspension in the cryotube has melted, take the cryotube into the clean bench, gently open the cryotube cover, use a 1mL pipette to transfer the cell suspension in the cryotube 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 blow to mix; transfer 1mL of cell suspension to a T25 culture flask containing 5mL DMEM complete medium, and shake slowly to make the cells evenly distributed; finally, place the culture flask in a 37℃, 5% CO2 incubator for culture, and replace the culture medium after the cells adhere to the wall;

[0062] Passaging: Take out the culture flask from the incubator and observe the cell growth under a microscope. When the cell coverage rate in the culture flask reaches more than 80%, discard the culture medium and wash once with 1×PBS buffer; then add about 1mL 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 2-3mL of DMEM complete culture medium to terminate the digestion; finally, use a 1mL pipette to blow the cell adherent surface to suspend all the adherent cells into a cell suspension; place the cell suspension in a centrifuge tube and centrifuge at 1000rpm for 5min; after centrifugation, discard the supernatant, add 2mL of DMEM complete culture medium and gently blow to mix, then divide the cell suspension into two and inoculate them into new culture flasks to complete the passaging;

[0063] Cryopreservation: After digesting the cells as described above, place the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 5 min; discard the culture medium, and resuspend the cells with cell freezing solution (50% complete culture medium + 40% FBS + 10% DMSO), and then divide them into cryopreservation tubes; let the cryopreservation tubes stand for a few minutes and then place them in a gradient cooling box, place them in a -150°C refrigerator for one day, and then transfer them to a cryopreservation box for long-term storage.

[0064] (2) Effects of two peptides on HepG2 cell viability

[0065] Cells were plated at 1×10 4 The cells were inoculated into 96-well plates 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 h (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 h until the liquid turned orange-yellow. The absorbance value was detected at a wavelength of 450 nm using an enzyme reader. The results are shown in Figure 2. Figure 6 shown.

[0066] 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 when the concentration of LPLLLPR was 2.5 and 5 μg / mL, it 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).

[0067] (3) Effects of two peptides on glucose uptake in IR HepG2 cells

[0068] The effects of LPLLLPR and ELILDIK on glucose consumption in the IR HepG2 cell model were investigated.

[0069] Cells were plated at 1×10 4 The cells were seeded into 96-well plates at a concentration of 10 μg / mL and pre-incubated overnight. After pre-incubation, HepG2 cells were exposed to -6 M insulin, cells not treated with insulin were used as the control group. After 36 hours of incubation, the cells were exposed or not exposed to the drug and incubated for 24 hours to detect the glucose content in the supernatant. The high glucose concentration in each group of cells was 22.5mmol / L. The final supernatant glucose content was measured by the glucose oxidase-peroxidase coupling method (GOD-POD). The specific operation method refers to the instructions of the glucose kit. The results are shown in Figure 7 Glucose consumption is calculated as follows:

[0070] Glucose consumption = glucose content of blank medium - glucose content of test group

[0071] Figure 7 The results showed that compared with the blank group, the glucose consumption of 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 could significantly increase the glucose consumption of IRHepG2 cells (P<0.05), and as the concentration increased, the glucose consumption also increased. This showed that LPLLLPR and ELILDIK can effectively improve the insulin resistance of HepG2 cells induced by glucose.

[0072] In summary, the LPLLLPR and ELILDIK obtained in the present invention effectively inhibit PTP1B and have a 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.

[0073] Finally, after the LPLLLPR and / or ELILDIK of the present invention is prepared into corresponding medicines or edible products, it can also effectively inhibit the activity of protein tyrosine phospholipase 1B, and can also effectively consume the amount of glucose in the IR HepG2 cell model, and has no cytotoxicity.

[0074] 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 protection scope of the present invention.

Claims

1. Application of camel milk active peptides in the preparation of edible products.

2. Application of camel milk active peptides in drugs for the treatment and / or prevention of diabetes.

3. Application of camel milk active peptides in the preparation of PTP1B inhibitors.

4. The use according to any one of claims 1 to 3, characterized in that: The camel milk active peptides include compound I with an amino acid sequence of LPLLLPR and / or compound II with an amino acid sequence of ELILDIK.

5. The use according to claim 4, characterized in that: The structure of the compound I is shown in Formula I:

6. The use according to claim 4, characterized in that: The structure of the compound II is shown in Formula II:

7. The use according to claim 4, 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.

8. The use according to claim 1, characterized in that: The edible product is selected from one of oral liquid, tea, lozenge, capsule, drink or effervescent tablet.

9. The use according to claim 2, characterized in that: The medicament includes one or more pharmaceutically acceptable excipients.

10. The use according to claim 2, 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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