Preparation method of grifola frondosa protease hydrolysate, blood pressure lowering polypeptide and application thereof

Abstract

The application discloses a preparation method of grifola frondosa protease hydrolysate and a blood pressure reducing polypeptide and application thereof. The sequence of the blood pressure reducing polypeptide is APPLRP, LPPLL, LPPLLL, LPPLPRP, LPPPLLK, LPPRLP, LPPRP or RPPLP, the blood pressure reducing polypeptide is an ACE targeted peptide, and is derived from grifola frondosa protease hydrolysate. The hexapeptide and heptapeptide with the blood pressure reducing function provided by the application can inhibit ACE activity, treat hypertension, are safe and easy to absorb, have no side effects, can be used for preparation or screening of antihypertensive drugs, and have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptides. Specifically, this invention relates to a method for preparing Grifola frondosa prote hydrolysate, its blood pressure-lowering peptide, and its applications. Background Technology

[0002] Grifola frondosa, belonging to the subphylum Basidiomycota, class Agaricomycetes, order Apophyceales, family Polyporaceae, and genus Grifola, is a type of mushroom used for both food and medicine. It is characterized by its high content of protein, polysaccharides, steroids, and phenols, and possesses effects such as lowering blood pressure, anti-oxidation, anti-cancer, lowering blood lipids, and lowering blood sugar.

[0003] Hypertension is a major risk factor for serious diseases such as cerebral infarction, myocardial infarction, kidney failure, and atherosclerosis. Therefore, preventing and controlling hypertension is key to preventing many diseases, especially cardiovascular diseases. Cardiovascular disease is the leading cause of death worldwide.

[0004] ACE is a blood pressure-related protease that plays a crucial role in the renin-angiotensin system (RAS). The RAS regulates blood pressure by modulating fluids within the body. ACE catalyzes the formation of angiotensin II (Ang II), which acts on venous and arterial muscles, inducing vasoconstriction. Ang II not only participates in blood pressure regulation but also primarily engages in vascular remodeling through the angiotensin type I receptor (AT1R). Vascular remodeling can further increase peripheral resistance, exacerbating hypertension. Captopril, enalapril, and other ACE inhibitors are commonly used as commercial drugs for the treatment of hypertension. However, they are often accompanied by side effects such as dry cough and shortness of breath. Therefore, the search for safer ACE inhibitors with minimal side effects has been a focus of attention. It is well known that ACE inhibitory peptides obtained from natural foods rarely cause adverse side effects and therefore have potential for further utilization. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems and shortcomings of the prior art and to provide a method for preparing Grifola frondosa prote hydrolysate, its blood pressure-lowering peptides, and their applications.

[0006] The objective of this invention is achieved through the following technical methods:

[0007] (I) A blood pressure-lowering polypeptide derived from grisaema protein hydrolysate

[0008] The blood pressure-lowering polypeptide is one of the following polypeptides:

[0009] Polypeptide A: APPLRP, with the amino acid sequence Ala-Pro-Pro-Leu-Arg-Pro, as shown in SEQ ID No. 1;

[0010] Polypeptide B: LPPLL, with the amino acid sequence Leu-Pro-Pro-Leu-Leu, as shown in SEQ ID No. 2;

[0011] Polypeptide C: LPPLLL, with the amino acid sequence Leu-Pro-Pro-Leu-Leu-Leu, as shown in SEQ ID No. 3;

[0012] Polypeptide D: LPPLPRP, with the amino acid sequence Leu-Pro-Pro-Leu-Pro-Arg-Pro, as shown in SEQ ID No. 4.

[0013] Polypeptide E: LPPPLLK, with the amino acid sequence Leu-Pro-Pro-Pro-Leu-Leu-Lys, as shown in SEQ ID No. 5;

[0014] Polypeptide F: LPPRLP, with the amino acid sequence Leu-Pro-Pro-Arg-Leu-Pro, as shown in SEQ ID No. 6;

[0015] Polypeptide G: LPPRP, with the amino acid sequence Leu-Pro-Pro-Arg-Pro, as shown in SEQ ID No. 7;

[0016] Polypeptide H: RPPLP, with the amino acid sequence Arg-Pro-Pro-Leu-Pro, as shown in SEQ ID No. 8.

[0017] The blood pressure-lowering polypeptide is an ACE-targeting peptide.

[0018] (II) A method for preparing grisaema protein hydrolysate

[0019] The Grifola frondosa prote hydrolysate includes one or more polypeptides selected from polypeptides A to H as described in claim 1.

[0020] The preparation method of the Grifola frondosa prote hydrolysate is as follows:

[0021] S1) Add the pollen of Grifola frondosa to water, adjust the pH, add alkaline protease, and after the enzymatic hydrolysis is completed, heat up to inactivate the alkaline protease. Then cool and centrifuge to collect the supernatant.

[0022] Step S1) specifically involves: adding Grifola frondosa pollen to water, adjusting the pH to 9.0–0.0, adding alkaline protease, and then performing enzymatic hydrolysis at a temperature of 45–50°C for 2–4 hours. After the enzymatic hydrolysis is completed, the temperature is raised to 90–100°C and maintained for 10–15 minutes. After cooling, the mixture is centrifuged, and the supernatant is collected.

[0023] The mass concentration of the maitake pollen in water is 3-10%, the amount of alkaline protease added is 0.6-1.0%, and the mesh size of the maitake pollen is 60-100 mesh.

[0024] S2) While stirring, slowly add anhydrous ethanol to the supernatant, then let it stand at 4-6°C for 12-24 hours, filter and collect the supernatant. Perform rotary evaporation on the supernatant to remove the ethanol. After ultrafiltration and drying, obtain the grisbane protein hydrolysate.

[0025] In step S2), the final mass concentration of anhydrous ethanol is 80%.

[0026] In step S2), ultrafiltration is performed using a filter membrane with a molecular weight cutoff of 1 to 3 kDa.

[0027] The drying methods include, but are not limited to, freeze-drying, spray drying, and vacuum drying.

[0028] (III) An ACE inhibitor

[0029] The ACE inhibitor includes the antihypertensive peptide as described in any one of claims 1 to 2, the maitake mushroom protein hydrolysate as described in claim 3, or the maitake mushroom protein hydrolysate obtained by the preparation method as described in any one of claims 4 to 8.

[0030] (iv) Application of the above-mentioned antihypertensive peptides, Grifola frondosa protein hydrolysate and ACE inhibitors

[0031] The blood pressure-lowering peptides, Grifola frondosa protease hydrolysate, and ACE inhibitors can be used for the preparation or screening of antihypertensive drugs.

[0032] The beneficial effects of this invention are as follows:

[0033] (1) The Grifola frondosa ACE inhibitory peptides APPLRP and LPPPRP provided in this invention have a significant inhibitory effect on ACE, IC50 50 The values ​​were 26.58 μM and 9.31 μM, respectively, which showed no obvious toxicity to zebrafish and had the effect of treating or improving hypertension.

[0034] (2) The present invention uses a controllable and environmentally friendly bio-enzymatic method to extract the protein hydrolysate of Grifola frondosa. By monitoring the enzymatic hydrolysis process, the hypotensive peptide of Grifola frondosa is released to the maximum extent. At the same time, the polysaccharide is removed by ethanol, which improves the utilization rate of raw materials. The preparation process is scientific and reasonable, simple to operate, and has strong industrial feasibility.

[0035] (3) The antihypertensive peptide provided by the present invention is obtained by enzymatic hydrolysis of Grifola frondosa. It is safe and has no toxic side effects. It has significant ACE inhibitory activity and has the effect of protecting vascular smooth muscle cells. Attached Figure Description

[0036] Figure 1 This is the mass spectrometry analysis result of polypeptide A;

[0037] Figure 2 This is the mass spectrometry analysis result of peptide D;

[0038] Figure 3 This is the structural formula of polypeptide A;

[0039] Figure 4 The structural formula of polypeptide D;

[0040] Figure 5 Figure showing the results of peptide A's resistance to intestinal digestion;

[0041] Figure 6 Figure showing the results of peptide D's resistance to intestinal digestion;

[0042] Figure 7 The diagram shows the docking results of peptide A and ACE molecules; (a) shows the overall spatial position of peptide A in ACE protein, (b) shows the spatial position of peptide A in ACE protein, (c) shows the 3D interaction diagram of peptide A and ACE protein, and (d) shows the 2D interaction diagram of peptide A and ACE protein.

[0043] Figure 8 The diagram shows the docking results of peptide D with ACE molecules; (a) shows the overall spatial position of peptide D in ACE protein, (b) shows the spatial position of peptide D in ACE protein, (c) shows the 3D interaction diagram of peptide A and ACE protein, and (d) shows the 2D interaction diagram of peptide A and ACE protein.

[0044] Figure 9 The effect of Grifola frondosa protein hydrolysate, polypeptide A, and polypeptide D on blood flow velocity is shown in the figure.

[0045] Figure 10 Figure 1 shows the effect of Grifola frondosa protein hydrolysate, polypeptide A, and polypeptide D on cardiac output.

[0046] Figure 11 Figure 1 shows the effect of Grifola frondosa protein hydrolysate, polypeptide A, and polypeptide D on blood vessel diameter.

[0047] Figure 12 Figure 1 shows the effect of Grifola frondosa protein hydrolysate, polypeptide A, and polypeptide D on blood vessel wall thickness.

[0048] Figure 13 The figure shows the effects of Grifola frondosa protein hydrolysate, polypeptide A, and polypeptide D on ACE and ACE2 mRNA expression. Detailed Implementation

[0049] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Unless otherwise specified, the reagents or instruments used are considered to be conventional products that can be purchased commercially.

[0050] Specific embodiments of the present invention are as follows:

[0051] (I) Preparation of Grifola frondosa protein hydrolysate

[0052] Example 1

[0053] Grifola frondosa pollen (60 mesh) was dispersed in distilled water at a ratio of 1:20 (w / v), and the pH of the solution was adjusted to 10.0 using 1M NaOH. Then, 1% alkaline protease (w / w) was added, mixed well, and incubated in a magnetic stirrer at a constant temperature of 45℃ for 4 hours. The degree of hydrolysis was measured during this period. After hydrolysis, the hydrolysate was boiled in a water bath for 10 minutes to inactivate any residual enzyme. After cooling to room temperature, the hydrolysate was centrifuged at 20℃ and 10000×g for 15 minutes, and the supernatant was collected. Anhydrous ethanol was slowly added to the supernatant until the final ethanol concentration reached 80%, with continuous stirring using a magnetic stirrer. After mixing, the mixture was allowed to stand at 4℃ for 12 hours. The precipitate was filtered off, and the supernatant was subjected to low-pressure rotary evaporation (45℃) to remove the ethanol until no alcohol odor remained. After filtration, ultrafiltration was performed using a 1 kDa membrane to obtain solutions with molecular weights >1 kDa and <1 kDa. The alkaline protease hydrolysate solution with molecular weight <1 kDa was then freeze-dried to obtain Grifola frondosa protease hydrolysate. This hydrolysate was used for subsequent evaluation of ACE inhibitory activity.

[0054] Calculations show that the degree of hydrolysis using alkaline protease is 47.35%.

[0055] Determination of degree of hydrolysis: The degree of hydrolysis of the Grifola frondosa enzymatic hydrolysate was determined using the pH-stat method. Formula for determining the degree of hydrolysis:

[0056]

[0057] In the formula:

[0058] V NaOH : Volume of NaOH consumed in the titration (mL);

[0059] M NaOH : Concentration of NaOH (M);

[0060] M P Protein mass (g);

[0061] H tot Peptide bond content (mmol / g), generally taken as 8.0;

[0062] α: Average degree of dissociation of amino acids.

[0063] Comparative Example 1

[0064] This comparative example uses Maitake pollen.

[0065] Comparative Example 2

[0066] In this comparative example, after enzymatic hydrolysis, the hydrolysate was inactivated, cooled, centrifuged, and the supernatant was collected without adding anhydrous ethanol. The remaining steps were the same as in Example 1 to obtain Grifola frondosa protein hydrolysate.

[0067] Comparative Example 3

[0068] In this comparative example, after enzymatic hydrolysis, the hydrolysate was inactivated, cooled, centrifuged, and the supernatant was collected. Anhydrous ethanol was slowly added to the supernatant until the final ethanol concentration was 60%. The remaining steps were the same as in Example 1 to obtain Grifola frondosa protein hydrolysate.

[0069] Comparative Example 4

[0070] In this comparative example, after enzymatic hydrolysis, the hydrolysate was inactivated, cooled, centrifuged, and the supernatant was collected. Anhydrous ethanol was slowly added to the supernatant until the final ethanol concentration was 70%. The remaining steps were the same as in Example 1 to obtain Grifola frondosa protein hydrolysate.

[0071] The polysaccharide content in the Grifola frondosa raw material / Grifola frondosa protein hydrolysate obtained in Example 1 and Comparative Examples 1 to 4 was measured respectively, and the results are shown in the table below. It can be seen that the addition of anhydrous ethanol removed the polysaccharides, thereby improving the utilization rate of the Grifola frondosa raw material.

[0072] Effect of different alcohol extraction concentrations on polysaccharide content

[0073]

[0074] (II) Isolation and Screening of Antihypertensive Peptides

[0075] The alkaline protease hydrolysate solution with a molecular weight <1kDa obtained in Example 1 was first desalted using a C18 desalting column, and then the sample was analyzed by LC-MS / MS equipped with an online nano-spray ion source. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC1200 in series. A total of 1 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient at a rate of 60 min. The column flow rate was controlled at 400 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, the gradient started from 0% B phase, increased to 60% nonlinearly at 46 min, increased to 100% within 4 min, and was maintained for 10 min.

[0076] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200-2000; Resolution: 70000; AGC target: 3e6; Maximum injection time: 60ms; (2) HCD-MS / MS: Resolution: 17500; AGC target: 5e4; Maximum injection time: 80ms; Collision energy: 27; Dynamic exclusion time: 20s.

[0077] Novel ACE inhibitory peptides were screened using an activity-based virtual screening tool, and their hydrophobicity, amphiphilicity, and other physicochemical properties were analyzed. The process included the following steps:

[0078] a) The activity of peptides obtained from virtual enzyme digestion was evaluated using the Peptide Ranker database (http: / / distilldeep.ucd.ie / PeptideRanker / ) to determine their potential for biological activity. Peptides with a score ≥ 0.85 were selected for further analysis.

[0079] b) The cell penetration ability of retrieved peptide sequences was predicted using the CPP pred (http: / / distilldeep.ucd.ie / CPPpred / ) database. Peptides with a score ≥ 0.5 were selected to screen for unreported ACE inhibitory peptides.

[0080] c) The potential biotoxicity and sensitization of peptide AH were assessed using AllerTOP v.2.0 (http: / / www.ddg-pharmfac.net / AllerTOP / ) and ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) online analysis software, and the physicochemical properties of each peptide, such as hydrophobicity, amphiphilicity, isoelectric point, and molecular weight, were analyzed.

[0081] The peptides obtained from the database screening were screened, and the in vitro ACE inhibitory activity was used as the evaluation index. The results are shown in Table 1 below.

[0082] Table 2. Bioinformatics-based prediction of Grifola frondosa peptide properties and in vitro ACE inhibitory activity

[0083]

[0084] Mass spectrometry results for peptide A and peptide D are shown in [reference needed]. Figure 1 and Figure 2 The amino acid sequence of polypeptide A is Ala-Pro-Pro-Leu-Arg-Pro (see...). Figure 3 ), its IC 50 The value was 26.58 μM; the amino acid sequence of peptide D was Leu-Pro-Pro-Leu-Pro-Arg-Pro (see...). Figure 4 ), its IC 50 The value was 9.31 μM. In vitro simulated digestion of peptides A and D was performed, and the results showed that both peptides A and D exhibited good digestibility (see...). Figure 5 and Figure 6 ).

[0085] In vitro ACE inhibitory activity evaluation: The reaction was performed in 0.1 M sodium borate buffer (pH 8.3, containing 0.3 M NaCl). The initial sample solution (50 μL) and ACE (50 μL, 0.1 U / mL) were pre-incubated at 37 °C for 5 min. Then, 50 μL of HCl (5 mM) was added, and the mixture was further incubated at 37 °C for 60 min. HCl (100 μL, 1 M) was added, followed by ultrapure water (100 μL). The mixture was filtered through a 0.45 μm microporous membrane, and 10 μL of the post-reaction solution was injected for analysis of the amount of HA generated.

[0086] Chromatographic conditions: Eclipse XDB-C18 column (4.6*250mm*5μm); mobile phase: acetonitrile-0.5% TFA (20:80); flow rate: 0.5mL / min; column temperature: 30℃; detection wavelength: 228nm.

[0087]

[0088] In the formula, A 空白 Indicates the peak area of ​​HA in the sodium borate buffer solution group; A 样品 This indicates the peak area of ​​HA in the sample group.

[0089] (III) ACE targeting of antihypertensive peptides

[0090] To further investigate the properties of the obtained peptides, the following samples were synthesized using the FOMC solid-phase synthesis method and used in subsequent experiments:

[0091] Polypeptide A: APPLRP;

[0092] Polypeptide B: LPPLL;

[0093] Polypeptide C: LPPLLL;

[0094] Polypeptide D: LPPRP;

[0095] Polypeptide E: LPPPLLK;

[0096] Polypeptide F: LPPRLP;

[0097] Peptide G: LPPRP;

[0098] Polypeptide H: RPPLP.

[0099] Global molecular docking was performed between peptides A and D and ACE. Molecular docking was implemented using the AutoDock 4.2.6 software package. The center coordinates of the docking box were set to the protein center to completely enclose the protein structure. The number of grid points in each XYZ direction was set to 50×50×50, the number of docking attempts was set to 100, and the remaining parameters used default values.

[0100] The binding energies of peptide A and peptide D to ACE were -7.567 and -5.895 kcal / mol, respectively, indicating that peptides A and D can stably bind to ACE. Based on the molecular docking results, the interaction between peptides and ACE was further analyzed.

[0101] The interaction forces of polypeptide A and polypeptide D with ACE are as follows: Figure 7 , Figure 8As shown in the figure, peptide A forms three hydrogen bonds (Arg114, Lys117, and Arg522), one salt bridge (Glu225), and hydrophobic interactions (Lys118, Tyr213, Glu403, Ser219, Glu123, Trp220, Met223, Asp121, Phe570, and Ser222) with ACE. Peptide D forms two hydrogen bonds (Asn586 and Thr226), one salt bridge (Glu225), and hydrophobic interactions (Pro573, Ser228, Arg253, Leu236, Glu239, Asp232, Met587, Ser588, Trp574, Pro585, Pro575, and Glu576) with ACE.

[0102] The results showed that peptide A formed more hydrogen bonds with ACE than peptide D. Hydrogen bonds are considered one of the most important non-covalent interactions in the binding process between ACE inhibitors and ACE. Analysis from the perspective of interaction forces also indicated that peptide A has a stronger affinity for ACE, consistent with the binding energy results.

[0103] (iv) Blood pressure lowering effects of Grifola frondosa protein hydrolysate and blood pressure lowering peptides

[0104] Wild-type AB strain zebrafish (3dpf) were randomly selected and cultured in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (1000 μg / mL) and the positive control valsartan (200 μg / mL) were administered in water. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received intravenous injection of angiotensin II (Ang II) to establish a zebrafish hypertension model. The normal control group consisted of untreated, normally cultured zebrafish, while the model control group consisted of zebrafish with hypertension induced by intravenous injection of Ang II.

[0105] After treatment at 28℃ for 2 days, 10 zebrafish were randomly selected from each experimental group to detect blood flow velocity, cardiac output, blood vessel diameter, blood vessel wall thickness, and mRNA expression levels of ACE and ACE2.

[0106] The results are as follows Figures 9-13 As shown in the figure. Experimental results showed that the enzymatic hydrolysate, peptide A, and peptide D could reduce cardiac output and blood flow velocity in the blood vessels of zebrafish in the model group, increase blood vessel diameter, improve blood vessel wall thickness, downregulate the expression of ACE mRNA, and upregulate the expression of ACE2 mRNA, all of which had therapeutic or ameliorative effects on hypertension.

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0108] The gene and protein sequences involved in this invention are as follows:

[0109] SEQ ID No.1;

[0110] Name: Polypeptide A

[0111] Source: Artificial Sequence

[0112] APPLRP

[0113] SEQ ID No.2;

[0114] Name: Polypeptide B

[0115] Source: Artificial Sequence

[0116] LPPLL

[0117] SEQ ID No. 3;

[0118] Name: Polypeptide C

[0119] Source: Artificial Sequence

[0120] LPPLLL

[0121] Name: Polypeptide D

[0122] Source: Artificial Sequence

[0123] LPPLPRP

[0124] Name: Polypeptide E

[0125] Source: Artificial Sequence (LPPPLLK)

[0126] Name: Polypeptide F

[0127] Source: Artificial Sequence (LPPRLP)

[0128] Name: Polypeptide G

[0129] Source: Artificial Sequence (LPPRP)

[0130] Name: Polypeptide H

[0131] Source: Artificial Sequence RPPLP.

Claims

1. A blood pressure-lowering polypeptide derived from Grifola frondosa prote hydrolysate, characterized in that: The blood pressure-lowering polypeptide is polypeptide A, and its amino acid sequence is shown in SEQ ID No.

1.

2. The antihypertensive polypeptide derived from Grifola frondosa prote hydrolysate according to claim 1, characterized in that: The blood pressure-lowering polypeptide is an ACE-targeting peptide.

3. A Grifola frondosa protein hydrolysate, characterized in that: Includes polypeptide A as described in claim 1.

4. An ACE inhibitor, characterized in that: It includes the blood pressure-lowering polypeptide as described in any one of claims 1 to 2, and the Grifola frondosa prote hydrolysate as described in claim 3.

5. The application of a blood pressure-lowering polypeptide as described in any one of claims 1-2, the Grifola frondosa prote hydrolysate as described in claim 3, or the ACE inhibitor as described in claim 4, characterized in that: Used for the preparation or screening of antihypertensive drugs.

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