Highland barley oligopeptides for the treatment of metabolic syndrome

Abstract

This application provides a highland barley oligopeptide (DYWSP) for the treatment of metabolic syndrome. This oligopeptide has strong antioxidant capacity, can significantly inhibit the activity of pancreatic lipase and angiotensin-converting enzyme, and has excellent performance in terms of toxicity, digestibility, and allergy resistance, showing potential for treating metabolic diseases such as metabolic syndrome.

Description

Technical Field

[0001] The present application belongs to the field of treatment of metabolic-related diseases. Specifically, the present application provides highland barley oligopeptides for treating metabolic syndrome. Background Art

[0002] With the changes in people's living standards and lifestyles, such as long-term sitting and long-term unhealthy eating patterns (excessive intake of high-fat, high-sugar, high-salt foods, etc.), metabolic syndrome has become increasingly prevalent worldwide in recent years. Metabolic syndrome is a collection of risk factors involving a variety of metabolic abnormalities including central obesity, hyperlipidemia, hypertension, hyperglycemia, and insulin resistance. The risk of death in patients with metabolic syndrome is twice that of the general population, and the risk of heart disease or stroke is three times that of the general population. In addition, metabolic syndrome is also associated with non-alcoholic fatty liver disease, osteoporosis, hyperuricemia, chronic kidney disease, and polycystic ovary syndrome. Therefore, metabolic syndrome has been nicknamed "the prelude to death" and "the hotbed of chronic disease."

[0003] Based on the results of epidemiological analysis in my country, the Chinese Medical Association proposed the diagnostic criteria for metabolic syndrome in 2013: (1) abdominal obesity: waist circumference ≥90cm for men and ≥85cm for women; (2) fasting HDL-C <40mg / dL; (3) fasting TG ≥150mg / dL; (4) hyperglycemia: fasting blood glucose ≥110mg / dL or blood glucose ≥140mg / dL 2 hours after glucose load or patients diagnosed with diabetes and receiving treatment; (5) blood pressure ≥130 / 85mmHg or patients diagnosed with hypertension and receiving treatment. Patients with three or more of the above criteria can be diagnosed with metabolic syndrome. Currently, the treatment of metabolic syndrome must first address various risk factors such as obesity, dyslipidemia, and hypertension in a timely manner to bring the relevant indicators back to normal range. Dietary lipids are first hydrolyzed by pancreatic lipase into fatty acids and monoglycerides, then form micelles with cholesterol, bile salts, etc., and finally absorbed by cells in the intestine. Therefore, effectively inhibiting pancreatic lipase activity can reduce the body's absorption of lipids and alleviate obesity and dyslipidemia. Angiotensin-converting enzyme (ACE) is a membrane-bound dipeptide carboxylase that is widely distributed in various human tissues and blood. It plays an important role in regulating blood pressure in the renin-angiotensin system and the kallikrein-bradykinin system. Currently, there are a variety of drugs that regulate lipid metabolism and lower blood pressure in clinical practice, such as simvastatin, clofibrate, orlistat, and captopril. These drugs are fast-acting and effective in controlling blood lipids and blood pressure, but they all have certain side effects, such as diarrhea, gastrointestinal bloating, and hepatotoxicity.

[0004] Based on the traditional Chinese medical theory of "medicine and food have the same origin", the use of functional ingredients from dietary sources to prevent and treat metabolic syndrome has significant scientific significance and social benefits. Barley is the crop with the largest planting area and the highest yield in Tibet. In 2020, Tibet's barley production was 795,000 tons, accounting for about 76% of the total grain output. Barley is rich in protein (9.29% to 13.79%), and studies have found that enzymatic hydrolysis of barley protein can produce bioactive peptides with various health benefits. The molecular weight of bioactive peptides is between that of proteins and amino acids. They have the advantages of low toxicity, no side effects, structural diversity, good biocompatibility, and easy absorption and utilization by the human body. Therefore, bioactive peptides have great application prospects in the production of functional foods, health foods and medicines. Summary of the Invention

[0005] The present application has obtained a highland barley oligopeptide of aspartic acid-tyrosine-tryptophan-serine-proline (Asp-Tyr-Trp-Ser-Pro, DYWSP) through extensive screening. It is natural, non-toxic, non-allergenic, non-carcinogenic, has a small molecular weight, good hydrophilicity, is difficult to digest, and is easily absorbed by the human body. The oligopeptide has significant DPPH free radical scavenging activity and a strong inhibitory effect on pancreatic lipase and angiotensin-converting enzyme. It is further found that it binds to the amino acid residues of pancreatic lipase and angiotensin-converting enzyme through hydrophobic interactions, salt bridges, hydrogen bonds, and π-π stacking to achieve the inhibitory activity.

[0006] On the one hand, the present application provides a highland barley oligopeptide for treating metabolic syndrome, wherein the amino acid sequence of the oligopeptide is DYWSP.

[0007] Furthermore, the oligopeptide has antioxidant activity.

[0008] Furthermore, the oligopeptide can be used to prepare health products with antioxidant function.

[0009] Furthermore, the oligopeptide has pancreatic lipase inhibitory effects and angiotensin converting enzyme inhibitory effects.

[0010] Furthermore, the oligopeptide can be used to prepare a pharmaceutical composition for treating metabolic syndrome.

[0011] On the other hand, the present application provides the use of the above oligopeptides in the preparation of pancreatic lipase inhibitors and / or angiotensin-converting enzyme inhibitors.

[0012] On the other hand, the present application provides a pharmaceutical composition for treating metabolic syndrome, which comprises the above-mentioned oligopeptide.

[0013] Furthermore, the pharmaceutical composition is an oral dosage form.

[0014] Furthermore, the pharmaceutical composition is in the form of tablets, capsules or oral liquids.

[0015] On the other hand, the present application provides a method for preparing the above oligopeptide, which is an Fmoc solid-phase synthesis method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the mass spectrometry result of peptide DYWSP;

[0017] Figure 2 To evaluate the activity effect of peptide DYWSP;

[0018] Figure 3 This is the molecular docking result diagram of peptide DYWSP and pancreatic lipase;

[0019] Figure 4 This is the molecular docking result of peptide DYWSP and angiotensin-converting enzyme. DETAILED DESCRIPTION

[0020] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and in no way limit the scope of protection of the present invention.

[0021] Example 1 Preparation and identification of highland barley oligopeptides

[0022] Defatted barley flour

[0023] Highland barley flour and n-hexane were mixed in a mass-to-volume ratio of 1:5, stirred continuously in a 37°C water bath with a constant temperature oscillator for 4 hours, allowed to stand for 1 hour to allow the highland barley flour to settle, and the upper n-hexane layer was poured out for recovery. Finally, the defatted highland barley flour was air-dried in a fume hood for 12 hours before use.

[0024] Separation of highland barley protein by alkali dissolution and acid precipitation

[0025] Defatted highland barley flour was mixed with distilled water at a mass-to-volume ratio of 1:25, and the pH of the solution was adjusted to 11 using 1 mol / L NaOH. After continuous stirring in a 60°C waterbath in a constant-temperature oscillator for 30 minutes, the mixture was centrifuged at 7000×g for 10 minutes at 4°C, and the supernatant was collected. The pH of the supernatant was adjusted to 4.5 using 1 mol / L HCl, and the protein was precipitated by centrifugation at 7000×g for 10 minutes at 4°C. The protein precipitate was collected, washed three times with distilled water, and then redissolved in distilled water. The pH was adjusted to 7.0 using 1 mol / L NaOH, and the mixture was freeze-dried and stored at -20°C.

[0026] Preparation of crude peptides by enzymatic hydrolysis

[0027] Highland barley protein was mixed with 35 mM sodium phosphate buffer (pH 6.2) at a mass-to-volume ratio of 1:20. The pH of the solution was adjusted to 7.0 using 1 mol / L NaOH. 5% alkaline protease (w / w) was added, mixed, and then enzymatically digested in a shaking water bath for 2 hours at 50°C. After completion of the enzymatic digestion, the hydrolyzate was incubated in a boiling water bath for 10 minutes to inactivate any residual enzyme. After cooling to room temperature, the hydrolyzate was centrifuged at 10,000 × g for 15 minutes at 4°C, and the supernatant was collected to obtain the crude peptide.

[0028] Preparation and identification of crude peptide <3 kDa fraction

[0029] Crude peptides are a complex composition, including peptides of varying molecular weights, free amino acids, and incompletely hydrolyzed proteins. To obtain bioactive peptides with clear sequence characteristics and accurately assess their structure-activity relationships, effective separation and purification of crude peptides is essential. Transfer 12 mL of highland barley protein hydrolysate to a 3 kDa centrifugal ultrafiltration tube and centrifuge at 5000 × g for 30 minutes at 4°C to obtain the crude peptide <3 kDa fraction.

[0030] The crude peptide fraction <3 kDa was desalted using a C18 desalting column and analyzed by LC-MS / MS equipped with an online nanospray ion source. The system consisted of a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) coupled to an EASY-nanoLC 1200. 1 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm × 25 cm, and separated using a 60-min gradient at a flow rate of 400 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The mobile phases A and B consisted of 0.1% formic acid in water and 80% ACN containing 0.1% formic acid. The gradient started at 0% B and increased nonlinearly to 60% over 46 minutes, then to 100% over 4 minutes, where it was maintained for 10 minutes.

[0031] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200–2000; resolution: 70,000; AGC target: 3e 6 ; Maximum injection time: 60ms; (2) HCD-MS / MS: resolution: 17500; AGC target: 5e 4 ; Maximum injection time: 80ms; Collision energy: 27; Dynamic exclusion time: 20s.

[0032] Tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was uniprot-Hordeum_vulgare_subsp_vulgare (version 2023, 34528 entries), with a "none" digestion setting. The search parameters included a fragment ion mass tolerance of 0.02 Da and a precursor ion mass tolerance of 10 ppm. The peptide card value was set to -101 gP ≥ 20. Peptides not found in the database were identified by setting an ALC (%) ≥ 80. Some representative results are shown in Table 1.

[0033] Bioinformatics-based screening of active peptides

[0034] Non-toxicity, non-allergenicity, non-carcinogenicity, and high potential bioactivity are the fundamental requirements for bioactive peptide screening. Small peptides with 2-10 amino acid residues and a molecular weight below 1000 Da can avoid gastrointestinal digestion, overcoming the drawback of proteins being broken down by digestive enzymes and thus unable to be taken orally. Therefore, using bioinformatics techniques, peptides in the <3 kDa fraction were initially screened for novel peptides that met development requirements based on criteria such as non-toxicity, non-allergenicity, non-carcinogenicity, high potential bioactivity, good hydrophilicity, poor gastrointestinal digestibility, and easy absorption by the human intestine.

[0035] Toxicity prediction for peptides was performed using the ToxinPrep (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) platform, based on the Support Vector Machine (Swiss-Port) algorithm. Allergenicity prediction was performed using AllerTOP v.2 (https: / / www.ddg-pharmfac.net / ddg / index.html). Carcinogenicity and intestinal absorption were predicted using admetSAR (http: / / lmmd.ecust.edu.cn / admetsar1 / home / ). The potential biological activity of peptides was analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd.ie / PeptideRanker / ), where a threshold greater than 0.5 was considered bioactive. Gastrointestinal digestibility of peptides was predicted using pepsin and trypsin using PeptideCutter (https: / / web.expasy.org / peptide_cutter / ). Overall average hydrophilicity was assessed using ExPasy (https: / / web.expasy.org / protparam / ). The overall average hydrophilicity can be used to characterize the hydrophilicity and hydrophobicity of a protein, with more negative values ​​indicating stronger hydrophilicity. Ultimately, based on the criteria of small molecular weight (<1000 Da), safety, high bioactivity (>0.5), good hydrophilicity, indigestibility, and easy absorption in the human intestine, the previously unreported peptide DYWSP was screened from highland barley protein for the first time (see Table 1).

[0036] Table 1 Prediction of highland barley peptide properties based on bioinformatics

[0037]

[0038]

[0039] Example 2 Synthesis, Activity Identification and Mechanism Analysis of Highland Barley Oligopeptides

[0040] The peptide DYWSP was prepared by Fmoc solid phase synthesis as follows:

[0041] Solvent treatment

[0042] N,N-dimethylformamide (DMF) and methanol were soaked with G3 molecular sieves overnight before use to remove impurities and water.

[0043] The resin is fully swollen

[0044] Weigh 2.0 g of blank Wang resin into a clean, dry reaction tube, add 15 mL of DMF, and activate at room temperature for 30 min.

[0045] First amino acid

[0046] At room temperature, remove the solvent from the previous step by suction through a sand core. Add 1 mmol of a 5-fold molar excess of the first C-terminal amino acid, a 5-fold molar excess of DMAP, and a 5-fold molar excess of N,N-diisopropylcarbodiimide (DIC) in DMF as the solvent. Allow to react at room temperature for 3 hours. After the reaction is complete, wash the reaction 4-6 times with 5-6 mL of DMF each time. Then, add pyridine and acetic anhydride in a 1:1 volume ratio and allow to react for 30 minutes. After the reaction is complete, wash the reaction 4-6 times with 5-6 mL of DMF each time.

[0047] Removal of the Fmoc protecting group

[0048] Remove the solvent from the previous step by suction, add 10 mL of 20% piperidine DMF solution to the resin, stir under N2 for 10 min, filter out the solution, add 10 mL of 20% piperidine DMF solution, stir under N2 for 5 min, and filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0049] Ninhydrin detection removal effect

[0050] Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105℃-110℃ for 5 minutes. If it turns dark blue, it is a positive reaction, indicating that the removal is complete and the next step can be carried out. If it is colorless, it means that the protecting group is not completely removed, and the above deprotection operation needs to be repeated.

[0051] Connect the second amino acid and remove the Fmoc protecting group

[0052] Weigh a 3-fold molar excess of the second C-terminal amino acid, a 3-fold molar excess of HBTU, and a 3-fold molar excess of 1-hydroxybenzotriazole into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve the mixture. Then, add a 10-fold molar excess of N,N-diisopropylethylamine. Allow to react at room temperature for 40 minutes. Wash with DMF 4-6 times, 5-6 mL each time. A small amount of resin should be tested with ninhydrin to detect colorlessness. Then, add 10 mL of 20% piperidine in DMF to remove the Fmoc residue. Repeat this process twice, 10 minutes and 5 minutes respectively. Wash the mixture four times with DMF and twice with methanol, 5-6 mL each time. Remove a small amount of resin and test with ninhydrin. If the color is blue, proceed to the next step.

[0053] Repeat the previous step by analogy until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain.

[0054] Resin shedding and pure product separation detection

[0055] Finally, the product was cut with trifluoroacetic acid cutting solution (95% trifluoroacetic acid: 2% triisopropylsilane: 2% ethanedithiol: 1% H2O) for 2 hours, and the reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The cleavage solution was blown dry with nitrogen, precipitated with ether, centrifuged, and then washed with ether 3 to 5 times to obtain a white solid. After dissolving in pure water, it was desalted and purified by HPLC, and lyophilized to precipitate crystals.

[0056] Quality testing of oligopeptides

[0057] A small amount of sample was dissolved by ultrasonication and then analyzed on a high-performance liquid chromatograph. HPLC parameters were as follows: column: ZORBAX SB-C18, 4.6×250 mm, 5 μm; aqueous phase: 100% water with 0.1% trifluoroacetic acid; organic phase: 100% acetonitrile with 0.1% trifluoroacetic acid; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 220 nm.

[0058] Mass spectrometry parameters: the ion source is an electrospray ionization source (ESI source), the nebulizing gas flow rate: 1.5 L / min, CDL: -20.0 V, CDL temperature: 250°C, heating block temperature: 200°C, ion source voltage: +4.5 kV, detector voltage: 1.5 kV, mobile phase flow rate: 0.2 mL / min, mobile phase ratio: 50% H2O / 50% ACN.

[0059] Finally, the purity of peptide DYWSP was determined to be greater than 95% by HPLC and mass spectrometry analysis. Figure 1 .

[0060] Verification of the active effects of bioactive peptides

[0061] DPPH free radical scavenging rate

[0062] After incubating 50 μL of a 5 mg / mL DYWSP solution (distilled water was used as a control group) and 50 μL of a 0.2 mmol / L DPPH (dissolved in methanol) free radical solution at 37°C in the dark for 20 minutes, the absorbance was recorded at 517 nm using a microplate reader. The DPPH free radical scavenging rate was calculated as follows:

[0063]

[0064] Where: A: control absorbance; B: sample absorbance.

[0065] Pancreatic lipase activity inhibition assay

[0066] A 2.5 mg / mL pancreatic lipase solution was prepared using pH 7.3 phosphate buffer, and then centrifuged at 5500 rpm for 5 min to obtain the supernatant. p-Nitrophenylbutyrate was diluted to 10 mM using pH 7.3 phosphate buffer.

[0067] After incubating 50 μL of 4 mg / mL DYWSP solution (with an equal amount of distilled water as a control group), 40 μL of pancreatic lipase solution, and 20 μL of p-nitrophenylbutyrate solution at 37°C for 30 minutes, the absorbance was recorded at 405 nm using a microplate reader. The pancreatic lipase activity inhibition rate was calculated as follows:

[0068]

[0069] Where: A: control absorbance; B: control blank absorbance; C: sample absorbance; D: sample blank absorbance.

[0070] Angiotensin-converting enzyme activity inhibition rate

[0071] 15 μ L 5mg / mL DYWSP solution and 10 μ L angiotensin converting enzyme (100mU / mL) are hatched 10min at 37 ℃.Then the substrate solution that mixture and 50 μ L contain 5mM hippurylhistidylleucine, 100mM sodium borate buffer (pH8.3,300mM sodium chloride) is hatched 60min at 37 ℃, then add 75 μ L 1M hydrochloric acid, 150 μ L pyridine and 75 μ L benzenesulfonyl chloride.Subsequently solution is vortexed 1min and cooled immediately in ice bath.Finally microplate reader measures the amount of the hippuric acid that releases at 410nm place.

[0072]

[0073] Where: A: control absorbance; B: control blank absorbance; C: sample absorbance.

[0074] Figure 2 The test results showed that the DPPH free radical scavenging rate and the inhibition rates of pancreatic lipase and angiotensin-converting enzyme activity of peptide DYWSP were 72.21±1.41%, 41.12±0.71%, and 95.93±1.94%, respectively. This indicates that peptide DYWSP has significant antioxidant activity and has strong inhibitory activity against pancreatic lipase and angiotensin-converting enzyme, providing new resources for the development of foods, health products, and pharmaceuticals for the prevention or treatment of metabolic syndrome, and has broad market prospects.

[0075] Analysis of the activity mechanism of bioactive peptides

[0076] Using the screened bioactive peptide DYWSP as a ligand, pancreatic lipase and angiotensin-converting enzyme as receptors, molecular docking was used to further determine the prevention or treatment mechanism of the peptide DYWSP in metabolic syndrome.

[0077] The peptide DYWSP was used for molecular docking with pancreatic lipase to clarify its target. The crystal structure of pancreatic lipase (PDB ID: 1ETH) was obtained from the RCSB Protein Database (http: / / www.rcsb.org / ). Flexible docking of the peptide DYWSP with pancreatic lipase was performed using Dock 6.9, and energy evaluation was performed based on the Grid scoring function. Chain A of the pancreatic lipase molecule (comprising 448 amino acid residues) was retained for docking analysis, while cocrystallized molecules and other polypeptide chains were removed. The docking was centered on the pancreatic lipase active site (Ser153, Asp177, and His264) at coordinates X: 64.153, Y: 39.278, and Z: 127.241. The docking score of the peptide DYWSP with pancreatic lipase was -83.780 kcal / mol, with a van der Waals contribution of -84.165 kcal / mol and an electrostatic contribution of 0.385 kcal / mol. The docking score of peptide DYWSP with pancreatic lipase is lower than -70 kcal / mol, so pancreatic lipase is a potential target for metabolic syndrome. Molecular docking simulation Figure 3 The results showed that the binding of the peptide DYWSP to pancreatic lipase residues mainly relies on hydrophobic interactions, salt bridges, hydrogen bonds, and π-π stacking. Specifically, DYWSP forms hydrophobic interactions with amino acid residues (Phe78, Ile79, Val260, Ala261, Leu265), hydrogen bonds with amino acid residue (His152), salt bridges with amino acid residues (Asp80, Arg257), and π-π stacking with amino acid residue (His264). Previous studies have found that pancreatic lipase contains an active site with the acylase catalytic triad Ser153, Asp177, and His264, as well as substrate-binding residues Phe78, Ile79, His152, Phe216, Trp253, and Arg257. Therefore, the peptide DYWSP inhibits the activity of pancreatic lipase by binding to the active sites Phe78, Ile79, His152, and His264 through hydrophobic interactions, hydrogen bonds, and π-π stacking.

[0078] The crystal structure of angiotensin-converting enzyme (PDB ID: 1O8A) was obtained from the RCSB protein database (http: / / www.rcsb.org / ). The peptide DYWSP was flexibly docked with angiotensin-converting enzyme using Dock 6.9, and the energy evaluation was performed based on the Grid scoring function. 2+ and Cl -, remove water molecules and other ligands. Molecular docking was centered on the S1 active pocket of angiotensin-converting enzyme (Ala354, Glu384 and Tyr523), that is, the coordinates are X: 44.766, Y: 34.044, Z: 46.363. The results showed that the docking score of peptide DYWSP with angiotensin-converting enzyme was -97.615 kcal / mol, the van der Waals force contribution was -95.777 kcal / mol, and the electrostatic force contribution was -1.838 kcal / mol. Molecular docking simulation Figure 4 The results showed that the binding of the peptide DYWSP to angiotensin-converting enzyme residues mainly relies on hydrophobic interactions, salt bridges, hydrogen bonds, and π-π stacking. Specifically, DYWSP forms hydrophobic interactions with amino acid residues (Asp358, Phe391, Glu403, Pro407), salt bridges with amino acid residue (Glu403), hydrogen bonds with amino acid residues (Asp358, His387), and π-π stacking with amino acid residue (Phe570).

Claims

1. A highland barley oligopeptide having antioxidant activity, pancreatic lipase inhibitory effect and angiotensin-converting enzyme inhibitory effect, characterized in that: The amino acid sequence of the highland barley oligopeptide is DYWSP.

2. Use of the highland barley oligopeptide according to claim 1 in the preparation of health products that contribute to anti-oxidation.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the highland barley oligopeptide according to claim 1. The pharmaceutical composition according to claim 3 , wherein the pharmaceutical composition is in an oral dosage form. The pharmaceutical composition according to claim 4 , wherein the pharmaceutical composition is in the form of tablets, capsules or oral liquids.

6. The method for preparing the highland barley oligopeptide according to claim 1, characterized in that: The method is an Fmoc solid phase synthesis method.

Images