Pressure-controlled bovine bone collagen active peptide

Abstract

The invention discloses a pressure-controlled bovine bone collagen active peptide, and belongs to the field of food. A potential ACE inhibitory peptide is rapidly screened through a bioinformatics method, the ACE inhibitory activity of the bovine bone collagen peptide is verified in combination with an in-vitro experiment, IC50 values of the screened bovine bone collagen active peptides LGALPGF, VFPGLL and AAAALGPW to ACE are 32.07 mu M, 40.41 mu M and 77.68 mu M respectively, and the peptide with the sequence is proved to have a good antihypertensive function.

Description

Technical Field

[0001] The invention relates to a pressure-controlled bovine collagen active peptide, belonging to the field of food. Background Art

[0002] Bovine bone collagen peptides, as small molecule peptides, exhibit a variety of biological activities, including antibacterial, anti-inflammatory, antioxidant, and bone health promotion. They are commonly used as raw materials in the preparation of health foods, cosmetics, and other products, enjoying high market acceptance. Due to their typical amino acid composition of type I collagen, they are high in hydrophobic amino acids and rich in glycine, proline, and alanine. FU et al. reported that collagen peptides with the repeating sequence Gly-XY, where X is typically Pro, can act as effective ACE inhibitors. Furthermore, Zhang Min's research has shown that the presence of a hydrophobic pocket in ACE favors binding to hydrophobic amino acids. In particular, peptides with a terminal hydrophobic amino acid and proline are more likely to bind to the ACE active pocket, resulting in stronger ACE inhibitory activity. This suggests that bovine bone collagen peptides may possess excellent blood pressure and blood sugar control activities. As food-derived peptides, they offer a higher oral safety profile than antihypertensive and hypoglycemic drugs. However, due to the wide molecular weight distribution and complex amino acid sequence of bovine bone collagen peptides, accurate assessment of their primary efficacy is difficult, significantly hindering their production and application.

[0003] In view of the rich peptide segments and diverse amino acid sequences in bovine collagen peptides, it is of great significance to combine molecular bioinformatics technology to screen, identify and verify the peptide segments with ACE activity and blood pressure control activity. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a rapidly identified pressure-regulating bovine collagen active peptide. By integrating molecular docking technology, bioinformatics prediction and traditional separation and purification processes, efficient screening and verification of bovine collagen peptide activity are achieved. The screened peptide segments have good pressure-regulating activity.

[0005] The present invention provides a bovine collagen peptide that can be rapidly identified and controls pressure, comprising the following steps: first, the bovine collagen peptide is continuously hydrolyzed by pepsin-trypsin, and then separated and purified step by step by ultrafiltration and RP-HPLC, and then three potential ACE inhibitory peptides are obtained by peptide sequence identification and bioinformatics screening, whose peptide sequences are LGALPGF, VFPGLL and AAAALGPW. According to in vitro experiments, these three peptides have good ACE inhibitory activity, and the half-inhibitory concentrations for ACE are 32.07μM, 40.41μM and 77.68μM, respectively. Consistent with the bioinformatics prediction results, this peptide is the first bovine collagen peptide with good pressure-controlling activity discovered by the present invention.

[0006] The present invention provides an ACE inhibitory peptide, wherein the amino acid sequence of the ACE inhibitory peptide is LGALPGF (SEQ ID No. 1), VFPGLL (SEQ ID No. 2) or AAAALGPW (SEQ ID No. 3).

[0007] The present invention provides products containing the ACE inhibitory peptide, which include food, medicine or health care products.

[0008] In one embodiment, the drug contains the ACE inhibitory peptide, a drug carrier and / or a pharmaceutical excipient.

[0009] In one embodiment, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills, and oral liquids.

[0010] In one embodiment, the drug carrier is one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

[0011] In one embodiment, the food comprises meat products, aquatic products, dairy products, cereal products or fruit and vegetable products.

[0012] The present invention provides the use of the ACE inhibitory peptide in the preparation of antihypertensive drugs.

[0013] In one embodiment, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0014] In one embodiment, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills, and oral liquids.

[0015] In one embodiment, the drug carrier is one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

[0016] The present invention has the following beneficial effects:

[0017] The present invention discloses a bovine collagen active peptide LGALPGF, VFPGLL and AAAALGPW, which has an IC of ACE. 50 The values ​​were 32.07 μM, 40.41 μM and 77.68 μM, respectively, demonstrating that the peptide of this sequence has good antihypertensive function.

[0018] The pressure-regulating bovine collagen peptide described in this invention can be rapidly identified through molecular bioinformatics and in vitro experiments to possess a stronger pressure-regulating effect than other bovine collagen peptides. Furthermore, the identification of this peptide can guide the production process for bovine collagen peptides with pressure-regulating activity, providing a theoretical and practical basis for expanding the application of this bovine collagen peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Molecular docking diagrams of AAAALGPW, LGALPGF, and VFPGLL. A: AAAALGPW; B: LGALPGF; C: VFPGLL; a: 3D molecular docking diagram; b: 3D schematic diagram of the interaction site; c: 2D schematic diagram of the interaction mode.

[0020] Figure 2 The activity of the synthetic peptide was verified. A: ACE inhibitory activity of the synthetic peptide at 100 μmol / L; B: IC 50 value. DETAILED DESCRIPTION

[0021] The bovine collagen peptides involved in the following examples were purchased from Wuxi Peptide Valley Biotechnology Co., Ltd. in Wuxi; the pepsin used was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd. with an enzyme activity of 3000 U / mg; and the trypsin was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd. with an enzyme activity of 250 U / mg.

[0022] Example 1 Enzymatic Hydrolysis of Bovine Collagen Peptide

[0023] 1. Continuous hydrolysis of bovine collagen peptides by gastric-trypsin

[0024] Bovine collagen peptide was dissolved at a 3% (m / v) concentration, pepsin was added to a final concentration of 2000 U / mL, and hydrolysis was carried out at 37°C for 2 hours. After hydrolysis, the pH of the solution was readjusted to 7.0, and trypsin was added to a final concentration of 100 U / mL. Hydrolysis was continued at 37°C for 2 hours. After the reaction, the sample was boiled at 95°C to inactivate the enzyme, cooled, and centrifuged at 5000 rpm for 20 minutes. The supernatant was collected and freeze-dried.

[0025] 2. ACE Inhibitory Activity Assay

[0026] To the ELISA plate, 100 μL of 1 mol / L FAPGG (N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine) (dissolved in 50 mmol / L Tris-HCl buffer, pH 7.5, containing 0.3 mol / L NaCl) and 50 μL of sample solution were added sequentially. Finally, 50 μL of 60 mU / mL ACE (dissolved in borate buffer, pH 8.3, containing 0.3 mol / L NaCl) was added to initiate the reaction. The reaction was incubated at 37°C for 30 minutes, and the decrease in absorbance at 345 nm was measured. A blank control was assayed using 50 mmol / L Tris-HCl buffer instead of the sample solution. The ACE solution was activated in a water bath at 37°C for 30 minutes before the reaction. The ACE inhibition rate was calculated as follows:

[0027]

[0028] Where: ΔA 空白 ΔA is the decrease in absorbance of the blank control group in 30 min; 样品 It is the decrease value of absorbance of the experimental group in 30 minutes.

[0029] 3. Isolation and purification of ACE inhibitory peptides

[0030] (1) Ultrafiltration

[0031] After simulated in vitro digestion, bovine collagen peptides were dissolved in deionized water and separated using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa. The ultrafiltration fractions with a molecular weight <2 kDa were collected, concentrated using a rotary evaporator, and then freeze-dried for use in the next purification experiment.

[0032] (2) RP-HPLC

[0033] Before separation and purification, the ultrafiltration collected fractions were dissolved in ultrapure water to prepare a solution with a protein concentration of 0.5 mg / mL, placed in a 100 Da dialysis bag for dialysis desalting, and then filtered and sterilized using a 0.22 μm pore size water microporous filter membrane. The purification process was completed using an XBridge Prep C18 preparative chromatography column (10 mm × 250 mm). The mobile phase system consisted of phase A (ultrapure water containing 0.1% trifluoroacetic acid) and phase B (acetonitrile containing 0.1% trifluoroacetic acid). The detection wavelength was set to 215 nm, the injection volume was 500 μL, and the elution flow rate was 2 mL / min. To ensure effective separation of the target components, a gradient elution procedure was used for separation and purification. The elution gradient was set as follows: 0-5 min, 90% A, 10% B; 5-10 min, 90%-40% A, 10%-60% B; 10-30 min, 40%-20% A, 60%-80% B; 30-35 min, 20% A, 80% B; 35-40 min, 20%-90% A, 80%-10% B. Fractions were collected according to peak concentration, concentrated by rotary evaporation, and then freeze-dried.

[0034] As shown in Table 1, after the raw bovine collagen peptide was continuously hydrolyzed by gastric-trypsin, its half-inhibitory concentration for ACE was 2.99 mg / mL. After that, a 2kDa ultrafiltration membrane was used to separate it, and after collecting the ultrafiltration retained fraction, it was found that the IC50 of bovine collagen peptide for ACE was 2.99 mg / mL. 50 The value decreased from 2.99 mg / mL to 1.92 mg / mL, and the half inhibition concentration decreased by 35.78%, indicating that ultrafiltration treatment significantly improved its inhibitory activity against ACE. The components collected by ultrafiltration were further purified by RP-HPLC, and three sub-components with different hydrophobicity (F1, F2 and F3) were obtained by peak collection. The three collected components were adjusted to a concentration of 1 mg / mL, and their inhibitory activity against ACE was determined. The results showed that the ACE inhibition rates of F2 and F3 components were higher than those of F1 component, among which the inhibition rate of F2 component was the highest, which was 55.97% ± 0.75%. The IC value of F2 component against ACE was also measured. 50 The value was 0.85 mg / mL. Compared with the fraction before RP-HPLC purification, its IC 50 The value decreased by 55.72%. This indicates that the ACE inhibitory activity of bovine collagen peptide was significantly improved after RP-HPLC purification. Further analysis showed that the increase in hydrophobicity contributes to the expression of the peptide's biological activity.

[0035] Table 1 Activity changes of bovine collagen peptides

[0036]

[0037] Example 2 Identification of bovine collagen ACE inhibitory peptides

[0038] The peptide sequence of the most potent ACE inhibitory component, obtained after two-step separation and purification, was identified using LC-MS / MS. The system consisted of an EASY-nanoLC 1200 nanoliter liquid chromatography system and an Orbitrap Fusion Lumos triple-in-one mass spectrometer. Chromatographic separation conditions were as follows: a PepMap C18 column (20 cm × 75 μm); mobile phase A consisted of ultrapure water containing 0.1% formic acid; and mobile phase B consisted of acetonitrile containing 0.1% formic acid. The chromatographic conditions were: injection volume 5 μL; column flow rate 300 nL / min; column temperature 40°C; and electrospray voltage 2 kV. A linear gradient elution was used: 96% to 50% A, 4% to 50% B, 0-53 min; 50% to 5% A, 50% to 95% B, 53-55 min; and 5% A, 95% B, 55-60 min. Mass spectrometry analysis was performed using data-dependent acquisition (DDA) mode, with automatic switching between full-scan MS and fragmentation MS / MS scans. Full-scan MS parameters were set as follows: mass scan range m / z 100–1500, resolution 120,000, and maximum injection time 100 ms. HCD-MS / MS fragmentation parameters were set as follows: resolution 50,000, maximum injection time 86 ms, with step-wise fragmentation at three collision energies of 25%, 30%, and 35%. A 30-s dynamic exclusion function was also enabled to avoid duplicate detection of highly abundant ions.

[0039] Bioinformatics screening

[0040] 1) Select peptide sequences with a confidence score of -101gP>50 and ALC>80% obtained by sample sequencing;

[0041] 2) The biological activity of the peptides was predicted on the Peptide Ranker website, and the bioactivity score was cut off at >0.8 points;

[0042] 3) Predict the hydrophobicity of peptides on the protein average hydrophobicity website, and cut off the hydrophobicity score > 0.5 points;

[0043] 4) Toxicity prediction of peptides was performed on the Toxin Pred website, and the toxicity score was cut off as <0;

[0044] 5) Analyze the homologous biological activities of peptides on the BIOPEP website;

[0045] 6) Perform molecular docking of the peptide with ACE to predict its docking binding energy, thereby predicting the potential ACE inhibitory activity of the peptide.

[0046] Peptide sequences from the F2 fraction were identified using LC / MS-MS, yielding 16,019 peptide sequences, including 1,969 database-searched peptides. Based on bioinformatics methods, peptides with identification scores of -10logP > 50 and ALC > 80% were selected and their bioactivity scores were predicted using the Peptide Ranker website. Ultimately, 326 peptides with bioactivity scores > 0.8 were screened. Further hydrophobicity prediction revealed 46 potentially bioactive peptides with good hydrophobicity. Toxicity prediction revealed negative support vector (SVM) values, indicating that these peptides were non-toxic. Subsequently, homologous bioactivity analysis using the BIOPEP website revealed that three of these peptides had previously been reported to exhibit similar bioactivity. Finally, these unreported, non-toxic, and hydrophobic peptides were subjected to 100 semi-flexible molecular dockings with ACE using the Auto Dock Tools software to estimate their docking binding energies. The lower the docking binding energy of the peptide with ACE, the greater the likelihood of its potent ACE inhibitory activity. Based on the relative molecular mass, biological activity, hydrophobicity score and ACE docking binding energy of the peptides, three peptides (Table 2) were finally selected for synthesis.

[0047] Table 2 Bioinformatics screening of ACE inhibitory peptides from bovine collagen

[0048]

[0049] To further analyze the interaction mechanism between peptides and ACE, the molecular docking results of LGALPGF, VFPGLL and AAAALGPW were visualized and analyzed using Pymol and Discovery Studio software ( Figure 1 AAAALGPW and LGALPGF both form hydrophobic interactions with His383, while AAAALGPW and VFPGLL form metal coordination interactions with Zn701. All three peptides form hydrogen bonds or hydrophobic interactions with Thr523 in the S1 pocket, Gln281 and His353 in the S2 pocket, and Glu162 in the S1' pocket.

[0050] Studies have shown that the antihypertensive drugs captopril and lisinopril can both bind to Zn701 in ACE and interact with the active sites His353 and Tyr520 through hydrogen bonds. In addition, lisinopril can also form hydrogen bond interactions with Glu162, Asp377, and Lys511, and form hydrophobic interactions with His383 and Val518. Related studies have shown that His353 is the key active site for the interaction between the ACE inhibitor lisinopril and ACE. The ACE inhibitory peptides discovered in this invention have some overlap in their mode of action with antihypertensive drugs, especially in their targeting of His353, a key site of action. Therefore, it is speculated that the reason these three polypeptides can express ACE inhibitory activity is that they can exert their inhibitory function by mimicking the key interaction between drug and receptor proteins.

[0051] Example 3 Synthesis and Activity Verification of Bovine Collagen ACE Inhibitory Peptide

[0052] The three peptides (AAAALGPW, LGALPGF and VFPGLL) obtained by bioinformatics screening in Example 2 were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and their inhibitory activities against ACE were verified respectively.

[0053] like Figure 2 As shown in the results, all three peptides showed certain ACE inhibitory activity at a concentration of 100 μmol / L, with the inhibition rates of AAAALGPW, LGALPGF, and VFPGLL being 55.56% ± 1.85%, 81.37% ± 0.98%, and 79.41% ± 2.94%, respectively. 50 The values ​​were 32.07μM, 40.41μM and 77.68μM respectively. 50 =44.84 μM) and FRW (IC 50 =46.30 μM), LGALPGF and VFPGLL have an IC 50 The values ​​are lower and significantly lower than those of the ACE inhibitory peptide CPF (IC 50 =131.35μM), RALP (IC 50 =650.00μM), TF (IC 50 =810.00 μM) and GHS (IC 50 =1740.00 μM). This indicates that the ACE inhibitory peptides discovered in this study all have good ACE inhibitory activity and can express strong blood pressure control activity.

[0054] In summary, through ultrafiltration and RP-HPLC step-by-step separation and purification, the half-inhibitory concentration of bovine collagen peptides for ACE was further reduced, and their inhibitory activity against ACE was continuously enhanced. The present invention successfully identified bovine collagen peptides LGALPGF, VFPGLL, and AAAALGPW with good ACE inhibitory activity, and the bovine collagen peptides were able to express good pressure-regulating activity.

[0055] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An ACE inhibitory peptide, characterized in that The amino acid sequence of the ACE inhibitory peptide is LGALPGF, VFPGLL or AAAALGPW.

2. A product containing the ACE inhibitory peptide according to claim 1, characterized in that: The products include food, medicine or health products.

3. The product according to claim 2, characterized in that The medicine contains the ACE inhibitory peptide according to claim 1, a drug carrier and / or a pharmaceutical excipient.

4. The product according to claim 3, characterized in that The dosage forms of the medicine include but are not limited to granules, capsules, tablets, pills, and oral liquids.

5. The product according to claim 4, characterized in that The drug carrier is one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

6. The product according to claim 2, characterized in that The food includes meat products, aquatic products, dairy products, cereal products or fruit and vegetable products.

7. Use of the ACE inhibitory peptide according to claim 1 in the preparation of antihypertensive drugs.

8. The use according to claim 7, characterized in that The medicine also contains a drug carrier and / or a pharmaceutical excipient.

9. The use according to claim 8, characterized in that The dosage form of the drug includes but is not limited to granules, capsules, tablets, pills, and oral liquid.

10. The use according to claim 9, characterized in that The drug carrier is one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent.

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