ACE (Angiotensin Converting Enzyme) inhibition and DPP-IV inhibition bifunctional peptide as well as design method and application thereof

Abstract

The invention discloses an ACE (Angiotensin Converting Enzyme) inhibition and DPP-IV (Dipeptidyl Peptidase-IV) inhibition bifunctional peptide as well as a design method and application thereof. The amino acid sequence of the bifunctional peptide fragment is Trp-Ala-Leu-Val-Ala-Pro or Trp-Pro-Leu-Val-Ala-Pro. According to the design method, hydrophobic amino acid replacement is carried out on most N-terminal first amino acid according to the characteristics of the DPP-IV inhibitory peptide, and N-terminal second amino acid is kept as proline Pro or alanine Ala. The difunctional peptide fragment obtained through the DPP-IV inhibitory activity optimization strategy has the effects of assisting in lowering blood pressure and lowering blood sugar at the same time and can be used for preparing functional food or health food with the functions of assisting in lowering blood pressure and lowering blood sugar, and the optimization strategy can be applied to improving the blood sugar lowering effect of the peptide fragment and has good market application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a DPP-IV inhibitory activity optimization strategy and application of a peptide segment. Background Art

[0002] Due to factors such as the development of the times, changes in lifestyle, urbanization, and an aging population, diabetes has become a global public health challenge, and its prevalence continues to rise. This chronic disease not only poses a threat to personal health, but also places a heavy burden on the global medical system. Hypertension is one of the common complications of diabetes. The coexistence of the two usually aggravates damage to the heart and blood vessels, leading to impaired cardiac function and heart failure. Although inhibitors with antihypertensive and hypoglycemic activities can effectively alleviate the effects of complications, synthetic angiotensin-converting enzyme (ACE) inhibitors and dipeptidyl peptidase (DPP)-IV inhibitors have been shown to cause adverse side effects, including hypotension, renal failure, liver failure, and decreased white blood cell counts. Therefore, there is an urgent need to develop natural bioactive substances with minimal side effects to treat hypertension and diabetes.

[0003] It is reported that bioactive peptides have a variety of biological activities, such as lowering blood pressure, lowering blood sugar, protecting the liver, and anti-oxidation. Due to their natural, safe, and easy processability, they can not only be used as part of daily supplements to promote health, but also have no side effects of drugs. In addition, bioactive peptides can treat hypertension and diabetes by inhibiting angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV). Bioactive peptides can be extracted not only from chicken, beef, and fish, but also from plants such as beans, broccoli, and dates. Among them, dates can be used as one of the sources of active peptides and are increasingly valued by people.

[0004] Jujube belongs to the genus Rhamnus and is rich in nutrients such as vitamins, amino acids, flavonoids, saponins, etc. It has high nutritional value and economic value. The health benefits of consuming red dates include anti-tumor, antioxidant, hypoglycemic, anti-inflammatory, hypotensive and liver protective effects. For example, the peptides IER and IGK were identified from jujube fruits and proved to have ACE inhibitory effects. In addition, we also identified the ACE inhibitory peptide KALVAP from the enzymatic products of jujube in previous studies, but its DPP-IV inhibitory activity was poor. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a DPP-IV inhibitory activity optimization strategy and application, and to modify and design the ACE inhibitory peptide KALVAP. The peptide segment obtained by the optimization of the present invention has both auxiliary blood pressure lowering and blood sugar lowering effects, and can be used to prepare functional foods or health foods with auxiliary blood pressure lowering and blood sugar lowering functions, which are particularly suitable for patients with diabetes accompanied by hypertension and have good market application prospects.

[0006] The technical solution adopted by the present invention to solve the above technical problems is:

[0007] A DPP-IV inhibitory activity optimization strategy was developed to modify the red jujube peptide Lys-Ala-Leu-Val-Ala-Pro (KALVAP) and design a peptide with enhanced DPP-IV inhibitory activity.

[0008] A DPP-IV inhibitory activity optimization strategy was developed. The database and literature of known DPP-IV inhibitory peptides were analyzed, the characteristics of DPP-IV inhibitory peptides were summarized, and KALVAP was modified and designed based on the summary.

[0009] A DPP-IV inhibitory activity optimization strategy, wherein the amino acid sequence of the DPP-IV inhibitory activity enhancing peptide is Trp-Ala-Leu-Val-Ala-Pro (WALVAP) or Trp-Pro-Leu-Val-Ala-Pro (WPLVAP).

[0010] A DPP-IV inhibitory activity optimization strategy, the modification method is to replace the first amino acid of most N-terminals with a hydrophobic amino acid to improve the DPP-IV activity of the peptide segment. The hydrophobic amino acid is tryptophan Trp, valine Val, leucine Leu or isoleucine Ile.

[0011] A DPP-IV inhibitory activity optimization strategy, wherein the modification method is to keep the second amino acid at the N-terminus as proline Pro or alanine Ala, which conforms to the characteristics of the DPP-IV enzyme substrate peptide segment.

[0012] A DPP-IV inhibitory activity optimization strategy, wherein the DPP-IV inhibitory peptide has the following characteristics:

[0013] (1) Peptides with highly hydrophobic amino acid residues generally have DPP-IV inhibitory activity;

[0014] (2) The first amino acid at the N-terminus is mostly a hydrophobic amino acid such as tryptophan Trp, valine Val, leucine Leu, isoleucine Ile, etc.;

[0015] (3) Proline (Pro) accounts for about 21.27% of the second amino acid at the N-terminus, and alanine accounts for about 9.41%. The presence of Pro or Ala at the second position of the peptide N-terminus and Leu or Ile at the N-terminus of the peptide is associated with relatively strong in vitro DPP-IV inhibitory activity;

[0016] (4) Peptides with Trp at the N-terminus usually show strong DPP-IV inhibitory activity;

[0017] (5) Peptides having Pro at the first, second, third or fourth N-terminal amino acid residue have strong DPP-IV inhibitory activity.

[0018] Application of a DPP-IV inhibitory activity optimization strategy to enhance the hypoglycemic effect of peptides.

[0019] A DPP-IV inhibitory activity optimization strategy is used in the preparation of auxiliary hypoglycemic health products and auxiliary hypoglycemic drugs.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] The present invention analyzes the amino acid sequences of existing DPP-IV inhibitory peptides, summarizes the amino acid sequence characteristics of DPP-IV inhibitory peptides, and based on this, KALVAP is modified and designed to obtain 11 peptides with potentially enhanced activity, synthesizes the peptides and screens the inhibitory activity, and verifies the cytotoxicity and DPP-IV inhibitory activity in a Caco-2 cell model, and the peptide sequences with improved DPP-IV inhibitory activity are Trp-Ala-Leu-Val-Ala-Pro (WALVAP) and Trp-Pro-Leu-Val-Ala-Pro (WPLVAP), and the in vitro DPP-IV IC 50 They were 57.6 and 102.5 μM, respectively. Compared with KALVAP, the DPP-IV inhibitory activity was improved both in vitro and in cells, indicating that the optimization strategy is feasible.

[0022] The DPP-IV inhibitory activity optimization strategy of the present invention, after the peptide KALVAP was modified and designed, it was found that the two peptides obtained had both ACE and DPP-IV inhibitory effects. At the same time, the inhibitory activity experiment verified that the peptides had both ACE and DPP-IV dual-target inhibitory effects. The ACE IC 50 20.5-35.2μM, DPP-IV IC 50The activity is 57.6-102.5 μM, and it is highly active. Compared with most of the current peptides that only have an ACE inhibitory function or a DPP-IV inhibitory function, the peptide of the present invention can simultaneously play an auxiliary role in lowering blood pressure and blood sugar, and has great advantages in preparing functional foods or health foods with auxiliary blood pressure and blood sugar lowering functions. This optimization strategy can be used to improve the blood sugar lowering effect of the peptide, and is particularly suitable for patients with diabetes and hypertension, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the molecular docking of peptides (A) KALVAP, (B) WALVAP, and (C) WPLVAP with DPP-IV;

[0024] Figure 2 Schematic diagram of the toxic effect of peptides on Caco-2 cells. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the accompanying drawings.

[0026] In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with the examples, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than to limit the claims of the present invention. The following examples are conducted using peptide segments synthesized by Shanghai Chupeptide Biotechnology Co., Ltd.

[0027] Experimental Example 1

[0028] Summary of the characteristics of DPP-IV inhibitory peptides and peptide optimization design

[0029] 1.1 Summary of the characteristics of DPP-IV inhibitory peptides:

[0030] An analysis of 489 DPP-IV inhibitory peptides and related literature in the BIOPEP database revealed the following characteristics:

[0031] (1) Peptides with highly hydrophobic amino acid residues generally have DPP-IV inhibitory activity;

[0032] (2) The first amino acid at the N-terminus is mostly a hydrophobic amino acid such as tryptophan Trp, valine Val, leucine Leu, isoleucine Ile, etc.;

[0033] (3) Proline (Pro) accounts for about 21.27% of the second amino acid at the N-terminus, and alanine accounts for about 9.41%. The presence of Pro or Ala at the second position of the peptide N-terminus and Leu or Ile at the N-terminus of the peptide is associated with relatively strong in vitro DPP-IV inhibitory activity;

[0034] (4) Peptides with Trp at the N-terminus usually show strong DPP-IV inhibitory activity;

[0035] (5) Peptides having Pro at the first, second, third or fourth N-terminal amino acid residue have strong DPP-IV inhibitory activity.

[0036] 1.2 Optimization design of peptides: According to the characteristics of DPP-IV inhibitory peptides, most of the first amino acids at the N-terminus were replaced with hydrophobic amino acids to improve the DPP-IV activity of the peptides. The second amino acid at the N-terminus was kept as proline or alanine, which is consistent with the characteristics of DPP-IV enzyme substrate peptides. Its characteristics are: ① The first amino acid Lys (K) and the second amino acid Ala (A) at the N-terminus in the peptide KALVAP sequence were replaced with Tyr (Y) and Pro (P), respectively, to obtain YPLVAP. ② The first amino acid Lys (K) at the N-terminus in the peptide KALVAP sequence was replaced with Tyr (Y), to obtain YALVAP. ③ The first amino acid Lys (K) at the N-terminus in the peptide KALVAP sequence was replaced with Trp (W), to obtain WALVAP. ④ The first amino acid Lys (K) and the second amino acid Ala (A) at the N-terminus in the peptide KALVAP sequence were replaced with Trp (W) and Pro (P), respectively, to obtain WPLVAP. ⑤ Replace the first amino acid Lys (K) and the second amino acid Ala (A) at the N-terminus of the peptide KALVAP sequence with Val (V) and Pro (P), respectively, to obtain VPLVAP. ⑥ Replace the first amino acid Lys (K) at the N-terminus of the peptide KALVAP sequence with Val (V), to obtain VALVAP. ⑦ Replace the second amino acid Ala (A) at the N-terminus of the peptide KALVAP sequence with Pro (P), to obtain KPLVAP. ⑧ Replace the first amino acid Lys (K) and the second amino acid Ala (A) at the N-terminus of the peptide KALVAP sequence with Ile (I) and Pro (P), respectively, to obtain IPLVAP. ⑨ Replace the first amino acid Lys (K) at the N-terminus of the peptide KALVAP sequence with Ile (I), to obtain IALVAP. HPLVAP was obtained by replacing the first amino acid Lys (K) and the second amino acid Ala (A) at the N-terminus of the peptide KALVAP sequence with His (H) and Pro (P), respectively.

[0037] Experimental Example 2

[0038] Determination of DPP-IV and ACE inhibitory activity of peptides

[0039] 2.1 Determination of DPP-IV inhibitory activity of peptide fragments:

[0040] The peptide was dissolved in pure water and prepared into a 100 μg / mL solution for preliminary screening of DPP-IV inhibitory activity. Peptides with an inhibition rate greater than 50% in the preliminary screening results were subjected to the next step of determination. The peptides that passed the preliminary screening were prepared into 5 sample peptide solutions of different concentrations. Take 50 μL of the peptide solution to a 96-well plate containing the reaction substrate Gly-Pro-p-nitroanilide (50 μL, 0.2 mM). Add 100 μL of DPP-Ⅳ solution (0.0025 U / mL). Incubate at 37°C for 60 minutes and read the absorbance at 405 nm. Tris-HCl buffer (0.1 M, pH 8.0) was used as a negative control and sitagliptin was used as a positive control. Each sample was analyzed in triplicate and the IC 50 The calculation formula of inhibition rate is as follows:

[0041] DPP-IV inhibition rate (%) = 1-(A 样品 -A 空白 / A 阴性对照 -A 空白 )×100

[0042] 2.2 Determination of ACE inhibitory activity of peptide fragments:

[0043] The peptide was dissolved in pure water, and 80 μl of 5 mM Hippuryl-histidyl-leucine (HHL) and 30 μl of the peptide solution were mixed in a centrifuge tube and incubated at 37°C for 5 min. 40 μl of 0.025 u / ml ACE solution (ACE was dissolved in HEPES buffer solution containing 300 mM NaCl and pH 8.3) was added, and the sample was further incubated at 37°C for 60 min. Then 150 μl / M HCl solution was added to terminate the reaction, and 700 μl of pure water was added. After mixing, the mixture was filtered through a 0.45 μm membrane and injected into an Agilent 1260 HPLC system (Santa Clara, CA, USA), which was equipped with CAPCELL PAK C18 AQ S-5 (10×150 mm; Shiseido, Tokyo, Japan). The drug captopril (10 ng / ml) was used as a positive control, and 30 μl of water was used instead of the peptide solution as a blank control. IC 50 Indicates the ability of hydrolyzate to inhibit ACE activity. All analyses were performed at 5 different concentrations and repeated 3 times. The inhibition rate is calculated as follows:

[0044] Inhibitory activity (%) = (HA 阳性 -HA 样品 ) / (HA 阳性 -HA 空白 )×100%

[0045] where HA positive, HA sample and HA blank are the relative areas of the HA peak.

[0046] Table 1 DPP-IV inhibition rate of peptides in Example 2, ACE IC 50 and DPP-IV IC 50

[0047]

[0048] Note: The DPP-IV inhibition rate here refers to the measurement when the peptide concentration is 100μg / mL.

[0049] Example 3

[0050] Molecular docking of peptides with DPP-IV

[0051] The structure of the purified peptide was constructed using ChemDraw Prime 19.1 software. Human dipeptidyl peptidase-IV (PDB code: 4A5S) was extracted from the RSCB protein database at a resolution of All water molecules and heteroatoms were deleted and hydrogen atoms were added. The docking was performed using Discovery Studio 2020 with a docking radius of Coordinates of DPP-IV: x=22.930y=27.759z=59.442.

[0052] Molecular docking of peptides (A) KALVAP, (B) WALVAP, and (C) WPLVAP with DPP-Ⅳ Figure 1 shown.

[0053] Table 2 Binding energy of peptides docked with DPP-IV receptor

[0054]

[0055] Experimental conclusion: Compared with KALVAP, the sites of action of WALVAP and WPLVAP with enhanced DPP-IV inhibitory activity on the DPP-IV receptor have undergone some changes: WALVAP has one less hydrogen bond with DPP-IV, and the different binding point is that the KALVAP active site Cys551 has changed to Ser209; WPLVAP has two less hydrogen bonds with DPP-IV, and the difference from the Arg358, Cys551 and Tyr585 binding sites of KALVAP is Arg125 and His126.

[0056] In addition, when the three peptides WALVAP, WPLVAP and KALVAP were docked with the DPP-IV receptor, KALVAP had the smallest docking energy and showed the best affinity, which was inconsistent with the actual measured DPP-IV inhibitory activity, indicating that the results of molecular docking should not only consider the size of the docking energy, but also focus on other factors such as the number of hydrogen bonds.

[0057] Example 4

[0058] In vitro gastrointestinal digestion

[0059] (1) According to the method of Hao et al., the samples were subjected to simulated intestinal digestion, and the simulated saliva (Simulated Salivary Fluid, SSF), simulated gastric fluid (Simulated Gastric Fluid, SGF) and simulated intestinal fluid (SIF) needed were prepared. The polypeptide was dissolved in pure water to prepare a sample solution with a concentration of 1 mg / ml.

[0060] (2) Oral stage: To 0.125 mL of sample, add 0.094 mL of SSF, 0.001 mL of CaCl2·2H2O (0.3 M), 0.025 mL of salivary amylase (11.25 mg / mL, 75 U / mL (final)), and 0.005 mL of H2O, totaling 0.25 mL, and incubate at 37°C, pH 7.0 for 2 min.

[0061] (3) Gastric stage: To 0.25 mL of oral digestive fluid, add 0.12 mL of SGF, 0.001 mL of CaCl2·2H2O (0.3 M), 0.048 mL of pepsin (6.94 mg / mL, 2000 U / mL (final)), 0.06 mL of gastric lipase (25 mg / mL, 60 U / mL (final)), 0.011 mL of H2O, and 0.01 mL of HCl (1 M), totaling 0.5 mL, and incubate at 37°C, pH 2.0 for 2 h.

[0062] (4) Intestinal stage: To 0.5 mL of gastric digestion fluid, add 0.195 mL of SIF, 0.001 mL of CaCl2·2H2O (0.3 M), 0.125 mL of pancreatic enzyme (3.2 mg / mL, 100 U / mL (final)), 0.1 mL of bile (40.857 mg / mL, 10 mM (final)), and 0.079 mL of H2O, totaling 1 mL, and incubate at 37°C, pH 7.0 for 2 h.

[0063] (5) Finally, the reaction was stopped with 150 μL of 1 M HCl.

[0064] Table 3 Molecular weight and in vitro gastrointestinal digestion stability of peptides

[0065]

[0066] Experimental conclusion: The final contents of the three peptides WALVAP, WPLVAP and KALVAP after digestion in a simulated gastrointestinal digestive environment in vitro were all above 90%, indicating that these three peptides have excellent digestion stability and can be used for subsequent development and utilization.

[0067] Example 5

[0068] Cell-based assay to verify DPP-IV inhibitory activity

[0069] 5.1 Cell culture of human colorectal cancer cells (Caco-2)

[0070] Caco-2 cells were cultured in DMEM medium containing 20% ​​fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 1% penicillin / streptomycin (P / S) at 37°C, 5% CO2 and 90% relative humidity, and passaged at a cell density of about 80%.

[0071] 5.2 Cytotoxicity of peptides on Caco-2 cells

[0072] Caco-2 cells in logarithmic growth phase were collected and 1×10 5 Cells were inoculated at a density of 100 μL / mL in a 96-well plate, with 100 μL of cell suspension in each well. After complete adherence, the samples were pretreated and blank and control groups were set up. After 24 hours of cell culture, 10 μL of CCK-8 reagent was added to each well. After further culture for 2 hours, the absorbance at 450 nm was measured using an ELISA reader. The cell survival rate was calculated as follows:

[0073] Cell survival rate = (experimental group - blank group) / (control group - blank group) × 100%

[0074] The toxic effects of peptides on Caco-2 cells Figure 2 shown.

[0075] Experimental conclusion: Under the action of WALVAP, WPLVAP and KALVAP, the cell survival rate was above 90%, which proved that the three peptides had no obvious cytotoxicity to Caco-2 cells and could be used to develop DPP-IV inhibitory type hypoglycemic drugs.

[0076] 5.3 DPP-IV inhibitory activity of peptides in Caco-2 cells

[0077] The cell suspension was diluted to a concentration of 2 × 10 5 / mL, 100μL was evenly inoculated in each well of a 96-well plate, and incubated for 24h until the cells adhered. After discarding the supernatant, the adherent cells were washed twice with PBS, 25μL of sample solution and 125μL of PBS were added, and the shake flask was incubated at 37℃ for 10min, and then 50μL of Gly-Pro-p-nitroanilide substrate (4mM) was added and the incubation continued. The absorbance was measured at 405nm, and the DPP-IV inhibitory activity was calculated.

[0078] Table 4 DPP-IV inhibitory activity of peptides in Caco-2 cells

[0079]

[0080] Experimental conclusion: WALVAP and WPLVAP peptides have significant inhibitory activity on DPP-IV enzyme in Caco-2 cells. 50 They were 138.1 and 142.5 μM, respectively. Compared with KALVAP, the DPP-IV inhibition rates of the two peptides WALVAP and WPLVAP were increased by 47.1 and 42.7 μM, respectively. In summary, we can conclude that this optimization scheme is feasible, and the modified WALVAP and WPLVAP have application prospects in the development of auxiliary hypoglycemic drugs.

Claims

1. A bifunctional peptide for ACE inhibition and DPP-IV inhibition, characterized in that: Its amino acid sequence is Trp-Ala-Leu-Val-Ala-Pro or Trp-Pro-Leu-Val-Ala-Pro.

2. A method for designing a bifunctional peptide for ACE inhibition and DPP-IV inhibition, characterized in that: The following steps are involved: The red date peptide segment Lys-Ala-Leu-Val-Ala-Pro was modified to design a bifunctional peptide with ACE inhibition and DPP-IV inhibition.

3. The method for designing a bifunctional peptide for ACE inhibition and DPP-IV inhibition according to claim 2, characterized in that: The Lys-Ala-Leu-Val-Ala-Pro red date peptide segment was modified, including: The first amino acid at the N-terminus of the red jujube peptide segment Lys-Ala-Leu-Val-Ala-Pro is replaced with a hydrophobic amino acid and the second amino acid at the N-terminus is kept as proline Pro or alanine Ala.

4. The method for designing a bifunctional peptide for ACE inhibition and DPP-IV inhibition according to claim 3, characterized in that: The hydrophobic amino acid is tryptophan Trp, valine Val, leucine Leu or isoleucine Ile.

5. The method for designing a bifunctional peptide for ACE inhibition and DPP-IV inhibition according to claim 1, characterized in that: The following steps are involved: The first amino acid at the N-terminus of the red jujube peptide Lys-Ala-Leu-Val-Ala-Pro was replaced with the hydrophobic amino acid tryptophan Trp and the second amino acid at the N-terminus was kept as proline Pro or alanine Ala, and a bifunctional peptide with ACE inhibition and DPP-IV inhibition was designed.

6. Use of the ACE inhibition and DPP-IV inhibition bifunctional peptide according to claim 1 in the preparation of auxiliary hypoglycemic health products and auxiliary hypoglycemic drugs.

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