Moss source of hypoglycemic peptide and its application

Abstract

The present application provides a Morchella source hypoglycemic peptide and application thereof, and belongs to the technical field of biological medicine. The present application provides a Morchella source hypoglycemic peptide, which comprises a tripeptide and / or a tetrapeptide; the amino acid sequence of the tripeptide is PTW, and the amino acid sequence of the tetrapeptide is MPTW. The Morchella source hypoglycemic peptide of the present application has a molecular weight less than 1000, strong hydrophobicity, and contains Pro and Trp. Molecular docking results show that PTW and / or MPTW can be combined with the proteinase diabetes prevention and treatment target dipeptidyl peptidase IV, protein tyrosine phosphatase 1B and alpha-amylase to play a hypoglycemic role through hydrogen bonding and hydrophobic interaction. Moreover, the hypoglycemic peptide of the present application is a natural product, has no hemolyticity and allergenicity, and has good pharmacokinetic characteristics. The present application has certain guiding significance for the development of food source hypoglycemic peptides.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a Morchella source hypoglycemic peptide and application thereof. BACKGROUND

[0002] Diabetes mellitus is a series of metabolic disorders of protein, fat and electrolyte caused by absolute or relative insulin secretion deficiency and decreased insulin sensitivity of target tissue cells. Diabetes mellitus has become a global public health problem. At present, the clinically used drugs (such as bosentan, repaglinide and repaglinide) can effectively control the blood glucose level of diabetes mellitus patients, but at the same time, a series of side effects (such as gastrointestinal diseases and diarrhea) will also be caused, therefore, searching for safe and less toxic natural active ingredients to prevent and treat diabetes mellitus has become a research hotspot in the field of food science and medicine in recent years.

[0003] Bioactive peptides have the advantages of simple structure, good safety, easy absorption and no immunoreactivity, not only have good nutritional and functional properties, but also have various health benefits such as antioxidant, blood pressure reduction and blood glucose reduction, and show good application prospects in the fields of functional food and medicine. At present, hundreds of hypoglycemic peptides have been isolated and prepared from different raw material protein hydrolysates, and food-derived hypoglycemic peptides are considered as a more healthy potential alternative for preventing and treating diabetes mellitus.

[0004] However, there is no report on Morchella source hypoglycemic peptides at present. SUMMARY

[0005] Therefore, the purpose of the present application is to provide Morchella source hypoglycemic peptides and application thereof, and the Morchella source hypoglycemic peptides of the present application have good blood glucose lowering activity.

[0006] The present application provides Morchella source hypoglycemic peptides, including tripeptides and / or tetrapeptides; the amino acid sequence of the tripeptide is PTW, and the amino acid sequence of the tetrapeptide is MPTW.

[0007] The present application also provides application of the Morchella source hypoglycemic peptides in the preparation of a blood glucose lowering product.

[0008] Preferably, the Morchella source hypoglycemic peptides can play a blood glucose lowering role by combining with proteinase diabetes prevention and treatment targets.

[0009] Preferably, the proteinase diabetes prevention and treatment targets include one or more of dipeptidyl peptidase IV, protein tyrosine phosphatase 1B and alpha-amylase.

[0010] The present application also provides a blood glucose lowering product, which comprises the Morchella source hypoglycemic peptides.

[0011] Preferably, the product comprises a pharmaceutical product.

[0012] Preferably, the dosage form of the pharmaceutical product comprises an oral preparation.

[0013] The present application also provides the use of the Morchella-derived hypoglycemic peptide described in the above-mentioned scheme in the preparation of an enzyme inhibitor; the enzyme comprises one or several of dipeptidyl peptidase IV, protein tyrosine phosphatase 1B and α-amylase.

[0014] The present application provides Morchella-derived hypoglycemic peptides, comprising tripeptides and / or tetrapeptides; the amino acid sequence of the tripeptide is PTW, and the amino acid sequence of the tetrapeptide is MPTW. The molecular weight of the Morchella-derived hypoglycemic peptides of the present application is less than 1000, and the peptides are strongly hydrophobic and contain Pro and Trp. Molecular docking results show that PTW and / or MPTW can bind to the key amino acid residues in the active center of the diabetes prevention and treatment target enzymes dipeptidyl peptidase IV (DPP-4), protein tyrosine phosphatase 1B (PTP1B) and α-amylase through hydrogen bonding and hydrophobic interaction to exert a hypoglycemic effect. The IC50 of PTW for DPP4, PTP1B and α-amylase is 16.08±1.11 mM, 5.89±0.21 mM and 18.75±0.43 mM, respectively; the IC50 of MPTW for DPP4, PTP1B and α-amylase is 6.34±0.03 mM, 3.06±0.12 mM and 14.59±0.17 mM, respectively. The hypoglycemic peptides of the present application belong to natural products, and have no hemolytic activity and allergenicity, and have good pharmacokinetic properties. The present application has certain guiding significance for the development of food-derived hypoglycemic peptides. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a mass spectrum of PTW;

[0016] Figure 2 is a mass spectrum of MPTW;

[0017] Figure 3 is a 2D diagram of the interaction between MPTW and DPP-4;

[0018] Figure 4 is a 2D diagram of the interaction between MPTW and PTP1B;

[0019] Figure 5 is a 2D diagram of the interaction between MPTW and α-amylase;

[0020] Figure 6 is a 3D diagram of the interaction between MPTW and DPP-4;

[0021] Figure 7 is a 3D diagram of the interaction between MPTW and PTP1B;

[0022] Figure 8 A 3D diagram showing the interaction between MPTW and α-amylase;

[0023] Figure 9 A 2D diagram of the interaction between PTW and DPP-4;

[0024] Figure 10 A 2D diagram of the interaction between PTW and PTP1B;

[0025] Figure 11 A 2D diagram showing the interaction between PTW and α-amylase;

[0026] Figure 12 A 3D diagram of the interaction between PTW and DPP-4;

[0027] Figure 13 A 3D diagram of the interaction between PTW and PTP1B;

[0028] Figure 14 A 3D diagram showing the interaction between PTW and α-amylase;

[0029] Figures 6-8 and Figures 12-14 The enzyme receptors are represented by rainbow-colored cartoons, and the peptide ligands are represented by yellow surfaces. Figures 3-5 as well as Figures 9-11 The green dashed lines represent hydrogen bonds. It represents hydrophobic properties. Detailed Implementation

[0030] This invention provides morel-derived hypoglycemic peptides, including tripeptides and / or tetrapeptides; the amino acid sequence of the tripeptide is PTW, and the amino acid sequence of the tetrapeptide is shown in SEQ ID NO.1, specifically MPTW.

[0031] In this invention, both PTW and MPTW have molecular weights less than 1000, poor solubility, strong hydrophobicity, zero net charge, and slightly acidic isoelectric points. Neither exhibits hemolytic or sensitizing properties, nor does it show significant aggregation or immunogenicity. Furthermore, the hypoglycemic peptides PTW and MPTW have different oral bioavailability, are readily absorbed by the intestine, and can cross Caco-2 cells and the blood-brain barrier to varying degrees. Their cytochrome P450 induction rate is less than 0.10, and their inhibition rate is less than 0.40, with both showing low human clearance rates, indicating strong metabolic stability. Both peptides exhibit virtually no Ames toxicity or Hek293 cytotoxicity. PTW and MPTW possess favorable pharmacokinetic characteristics.

[0032] In the present application, the PTW and MPTW are preferably obtained by solid-phase synthesis. In the implementation of the present application, the PTW and MPTW are obtained by solid-phase synthesis by Shanghai Synpeptide Biotech Co., Ltd.

[0033] The present application also provides the use of the Morchella-derived hypoglycemic peptide in the preparation of a product for lowering blood sugar.

[0034] In the present application, the Morchella-derived hypoglycemic peptide can exert a hypoglycemic effect by binding to a protease diabetes prevention and treatment target.

[0035] In the present application, the protease diabetes prevention and treatment target preferably includes one or more of DPP-4, PTP1B and alpha-amylase.

[0036] The present application also provides a product for lowering blood sugar, which includes the Morchella-derived hypoglycemic peptide described in the above scheme.

[0037] In the present application, the product preferably includes a pharmaceutical product.

[0038] In the present application, the dosage form of the pharmaceutical product preferably includes an oral preparation.

[0039] The present application also provides the use of the Morchella-derived hypoglycemic peptide described in the above scheme in the preparation of an enzyme inhibitor; the enzyme includes one or more of DPP-4, PTP1B and alpha-amylase.

[0040] In the present application, MPTW is combined with Glu205, His740 and Arg125 of DPP-4 by hydrogen bonding, and is combined with Asn545, Asn562, Tyr(48,547,631,662,752), Trp(563,627,629) and Ser630 by hydrophobic interaction; is combined with Glu200 of PTP1B by hydrogen bonding, and is combined with Leu192, Asn193, Phe(196,280), Lys197, Arg199, Gly277 and Glu276 by hydrophobic interaction; is combined with Thr163, Asp197 of alpha-amylase by hydrogen bonding, and is combined with Trp(58,59), Tyr62, Gln63, Arg195, Glu233, Phe256, Asn298, His(299,305) and Asp(300,356) by hydrophobic interaction.

[0041] PTW binds to Arg125, Glu206 and Tyr662 of DPP-4 by hydrogen bond, to Glu205, Tyr(547, 631, 666), Trp(629, 659), Val(656, 711) and Ser630 by hydrophobic interaction; to Glul 15, Trpl79, Argl21 and Gln262 of PTP1B by hydrogen bond, to Tyr46, Lysl20, Glyl83, Val184, Alal 17, Thr263, Asp265 and Gln266 by hydrophobic interaction; to Tyr62 and Asp300 of a-amylase by hydrogen bond, to Trp(58, 59), Gln63, Thr163, Leul65, Argl95, Asp197, Glu233, His(101, 299, 305) and Asp356 by hydrophobic interaction.

[0042] In the present application, the IC50 of PTW for DPP4, PTP1B and a-amylase inhibition is 16.08 ± 1.11 mM, 5.89 ± 0.21 mM and 18.75 ± 0.43 mM, respectively; the IC50 of MPTW for DPP4, PTP1B and a-amylase inhibition is 6.34 ± 0.03 mM, 3.06 ± 0.12 mM and 14.59 ± 0.17 mM, respectively. PTW and MPTW have good inhibitory effect on DPP-4, PTP1B and a-amylase.

[0043] The technical solutions in the present application will be described clearly and completely in combination with the examples in the present application.

[0044] Example 1 Screening process of Morchella-derived hypoglycemic peptides in the present application and their physicochemical properties

[0045] 154 Morchella major structural protein sequences were downloaded from UniProt KB protein database (https: / / www.uniprot.org / ). The sequences were optimized using CALCULATIONS module of BIOPEPUWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ). Specifically, the downloaded 154 Morchella protein sequences were pasted into the CALCULATIONS module of BIOPEPUWM database and analyzed, and the higher-scored 20 Morchella proteins were obtained as experimental research materials by comprehensively considering ΣA value (frequency of hypoglycemic peptides; set to 70%) and ΣB value (potential hypoglycemic activity; set to 30%).

[0046] The 20 Morchella proteins preliminarily screened were subjected to virtual enzymolysis through the "ENZYME(S) ACTION" module in the BIOPEP UPWM database. The 40S ribosomal protein S9 protein with the highest score was selected as the raw material protein for preparing Morchella-derived hypoglycemic peptides based on the ΣAE value (frequency of release of hypoglycemic peptides; 50% share) and the ΣW value (relative frequency of release of hypoglycemic peptides; 50% share) (Table 1).

[0047] Table 1 Selection of Morchella proteins and results of virtual enzymolysis

[0048]

[0049] The potential of the obtained peptide segments for biological activity was evaluated using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ). The peptide segments with a PeptideRanker score greater than 0.5 were subjected to a second round of screening using iDPPIV-SCM (http: / / camt.pythonanywhere.com / iDPPIV-SCM); in combination with novelty detection, two Morchella-derived hypoglycemic peptides PTW and MPTW with a high iDPPIV-SCM score and which had not been reported were screened (Table 2). The mass spectra of PTW and MPTW are shown in Figure 1 and Figure 2

[0050] Table 2 Two Morchella-derived hypoglycemic peptides obtained through virtual screening

[0051]

[0052] ​The molecular weight, isoelectric point and solubility of the hypoglycemic peptides were predicted by Innovagen (http: / / www.innovagen.com), the net charge and hydrophobicity were predicted by Pepdraw (http: / / www.tulane.edu / ~biochem / WW / PepDraw / ), the allergenicity was predicted by Allergylab (https: / / sortaller.gzhmu.edu.cn / ), the hemolyticity was predicted by HemoPI (https: / / webs.iiitd.edu.in / raghava / hemopi / design.php), the aggregation was predicted by ChemAGG (https: / / admet.scbdd.com / ChemAGG / index / ), and the immunogenicity was predicted by IEDB (http: / / tools.iedb.org / immunogenicity / ). The prediction results of the physicochemical properties of the two Morchella-derived hypoglycemic peptides of the present application are shown in Table 3.

[0053] Table 3 Physicochemical properties of Morchella-derived hypoglycemic peptides

[0054]

[0055] As can be seen from Table 3, the molecular weight of the Morchella-derived hypoglycemic peptides PTW and MPTW of the present application is less than 1000, the solubility is poor, the hydrophobicity is strong, the net charge is zero, and the isoelectric point is acidic; neither of them has hemolyticity and allergenicity, and basically has no aggregation and immunogenicity. The Morchella-derived hypoglycemic peptides of the present application have good physicochemical properties and safety.

[0056] Example 2 Pharmacokinetics of Morchella-derived hypoglycemic peptides

[0057] The pharmacokinetic properties of the hypoglycemic peptides of the present application were predicted by the iDrug (https: / / drug.ai.tencent.com / console / cn / admet) tool, and the results are shown in Table 4. It can be seen that the hypoglycemic peptides PTW and MPTW have different oral bioavailability, are easily absorbed by the intestinal tract, can pass through the Caco-2 cells and the blood-brain barrier to different degrees, have a cytochrome P450 induction rate less than 0.10, an inhibition rate less than 0.40, and a low human clearance rate, indicating that they have strong metabolic stability; the two peptides basically have no Ames toxicity and Hek293 cell toxicity. In summary, the Morchella-derived hypoglycemic peptides of the present application have good pharmacokinetic properties.

[0058] Table 4 Prediction results of pharmacokinetic properties of hypoglycemic peptides

[0059]

[0060] Note: The solubility coefficient is the thermodynamic water solubility value of the substance, expressed as Log(S, mol / L); Caco-2 cell permeability is the Caco-2 permeability value (10 mol / L). -6 cm / s); Human clearance rate reflects the excretion of the test substance in the human body and is expressed as log10 (ml / min / kg); Other indicators are represented by probability values ​​(between 0 and 1).

[0061] Example 3: Molecular docking of morel-derived hypoglycemic peptides

[0062] Molecular docking is a powerful tool for studying the interaction between peptides and target protein receptors. Using the HPEPDOCK tool (http: / / huanglab.phys.hust.edu.cn / hpepdock / ), with a hypoglycemic peptide as a ligand, docking studies were conducted with potential protease-based diabetes prevention and treatment targets (DPP-4, PTP1B, and α-amylase). The docking scores are shown in Table 5. Table 5 shows that MPTW achieved the highest docking scores with the three enzyme receptors, while PTW also had a relatively high docking score.

[0063] Table 5. Molecular docking results of morel-derived hypoglycemic peptides with DPP4, PTP1B, and α-amylase.

[0064]

[0065] The molecular docking results of MPTW with three enzyme receptors can be found in [link to documentation]. Figures 3-8 ,in Figure 3 , 4 Figures 5 and 6 show the 2D interaction diagrams of MPTW docking with DPP-4, PTP1B, and α-amylase, respectively. Figure 6 , 7 Figures 8 and 9 represent the 3D interaction diagrams of MPTW docking with DPP-4, PTP1B, and α-amylase, respectively. Figures 3-8This indicates that MPTW binds to Glu205, His740, and Arg125 of DPP-4 via hydrogen bonds, and to Asn545, Asn562, Tyr(48,547,631,662,752), Trp(563,627,629), and Ser630 via hydrophobic interactions; it binds to Glu200 of PTP1B via hydrogen bonds, and to Leu192, Asn193, and Phe(196) It binds to Trp(280), Lys197, Arg199, Gly277, and Glu276 via hydrophobic interactions; it binds to Thr163 and Asp197 of α-amylase via hydrogen bonds, and to Trp(58,59), Tyr62, Gln63, Arg195, Glu233, Phe256, Asn298, His(299,305), and Asp(300,356) via hydrophobic interactions.

[0066] See the molecular docking results of PTW with three enzyme receptors. Figures 9-14 ,in Figure 9 , 10 Figures 1 and 11 show the 2D interaction diagrams of PTW docking with DPP-4 PTP1B and α-amylase, respectively. Figure 12 , 13 Figures 1 and 14 show the 3D interaction diagrams of PTW with DPP-4, PTP1B, and α-amylase, respectively. Figures 9-14 This indicates that PTW binds to Arg125, Glu206, and Tyr662 of DPP-4 via hydrogen bonds, and to Glu205, Tyr(547,631,666), Trp(629,659), Val(656,711), and Ser630 via hydrophobic interactions; it also binds to Glu115, Trp179, Arg221, and Gln262 of PTP1B via hydrogen bonds, and to Tyr46, Lys120, ... Gly183, Val184, Ala217, Thr263, Asp265, and Gln266 bind with hydrophobic interactions; they bind with Tyr62 and Asp300 of α-amylases via hydrogen bonds, and with Trp(58,59), Gln63, Thr163, Leu165, Arg195, Asp197, Glu233, His(101,299,305), and Asp356 via hydrophobic interactions.

[0067] The morel-derived hypoglycemic peptide of this invention can bind to Glu (205,206), Tyr (547,631,662,666), Ser630, Val656, Trp659, Val711, and His740 of DPP-4, which are all key amino acid residues in the enzyme's active pocket. [1]Except for a few amino acids such as Trp179, Gly183, Val184, Gln266, and Asp265, all other amino acid residues of the morel-derived hypoglycemic peptide that bind to PTP1B are associated with Ali et al. [2] The reported results for the docking of phycoplasmidoquinone with PTP1B were consistent; furthermore, the morel-derived hypoglycemic peptides binding to α-amylase, including Trp(58,59), Tyr62, Gln63, Thr163, Arg195, Asp197, Glu233, His305, and Asp300, are all important residues in the active site of this enzyme. [3] .

[0068] Example 4: Hypoglycemic activity of morel-derived hypoglycemic peptides

[0069] In vitro chemical experimental model method [4-6] The inhibitory effects of PTW and MPTW on DPP-4, PTP1B, and α-amylase were evaluated (Table 6). It can be seen that PTW and MPTW all exhibited good inhibitory effects on DPP-4, PTP1B, and α-amylase.

[0070] Table 6. IC50 values ​​of PTW and MPTW for inhibiting DPP-4, PTP1B and α-amylase

[0071]

[0072] Note: The IC50 value of morel-derived hypoglycemic peptide is mM, the IC50 value of the positive control vildagliptin is nM, and the IC50 value of sodium orthovanadate and acarbose is μM.

[0073] References:

[0074] [1].Zhao L, Zhang M, Pan F, Li J, Dou R, Wang X, Wang Y, He Y, WangS, Cai S: In silico analysis of novel dipeptidyl peptidase-IV inhibitorypeptides released from Macadamia integrifolia antimicrobial protein 2(MiAMP2) and the possible pathways involved in diabetes protection. Current research in food science 2021, 4:603-611.

[0075] [2].Ali MY, Kim DH, Seong SH, et al. α-Glucosidase and proteintyrosine phosphatase 1B inhibitory activity of plastoquinones from marinebrown alga Sargassum serratifolium. Marine drugs 2017, 15(12):368.

[0076] [3].Khan M, Alam A, Khan KM, et al. Flurbiprofen derivatives as novelα-amylase inhibitors: Biology-oriented drug synthesis (BIODS), in vitro, andin silico evaluation. Bioorganic chemistry 2018, 81:157-167.

[0077] [4]. Yang Zhenzhen: Effects of three hypoglycemic plants in Xinjiang on dipeptidyl peptidase IV activity. Xinjiang Medical University; 2012.

[0078] [5]. Niu Enli: Design, synthesis and in vitro inhibitory activity study of sulfonamide protein tyrosine phosphatase 1B inhibitors. Beijing Institute of Technology; 2016.

[0079] [6].Cheng Q, Cai S, Ni D, Wang R, Zhou F, Ji B, Chen Y: In vitroantioxidant and pancreatic α-amylase inhibitory activity of isolated fractions from water extract of Qingzhuan tea. Journal of food science and technology 2015, 52(2):928-935.

[0080] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a Morchella hypoglycemic peptide in the preparation of a hypoglycemic drug, characterized in that, The Morchella-originated hypoglycemic peptide is a tripeptide or a tetrapeptide; the amino acid sequence of the tripeptide is PTW, and the amino acid sequence of the tetrapeptide is MPTW.

2. Use according to claim 1, characterized in that, The dosage form of the medicine includes oral preparations.

Images