A royal jelly-derived xo inhibitory peptide, and a method for obtaining and use thereof

Abstract

The application provides a royal jelly-derived XO inhibitory peptide as well as an obtaining method and application thereof, and relates to the technical fields of bioactive peptides and biomedical engineering. The amino acid sequence of the royal jelly-derived XO inhibitory peptide is selected from one or more of FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR and YYVNTEQFR; royal jelly is used as raw material, high hydrostatic pressure pretreatment is combined with continuous enzymolysis of pepsin and trypsin, and a peptide segment is identified by using liquid chromatography-tandem mass spectrometry to obtain multiple required inhibitory peptides. The application overcomes the shortcomings of the prior art, and provides a new approach for developing xanthine oxidase inhibitory products.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides and biomedical engineering technology, specifically to a royal jelly-derived XO inhibitory peptide, its acquisition method, and its application. Background Technology

[0002] Uric acid is the end product of purine metabolism in the human body. Increased uric acid production or decreased excretion can lead to elevated blood uric acid levels and is associated with the development of hyperuricemia, gout, and related metabolic abnormalities. Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine, which in turn catalyzes the formation of uric acid, making it a key enzyme in the uric acid production pathway. Therefore, inhibiting XO activity is an important technical approach to reduce uric acid production.

[0003] Currently, commonly used XO inhibitors include chemically synthesized drugs such as allopurinol. These drugs have a definite XO inhibitory effect, but some users may experience hypersensitivity reactions or other adverse reactions. Therefore, developing naturally derived peptides with well-defined structures, traceable origins, and XO inhibitory activity has research and application value.

[0004] Royal jelly is a substance secreted by the hypopharyngeal and mandibular glands of worker bees and is rich in protein. Major royal jelly proteins (MRJPs) are the main components of royal jelly protein and an important source of bioactive peptides. Previous studies have shown that MRJPs can release short peptides with various biological activities after enzymatic hydrolysis; however, systematic research on the specific sequences, preparation conditions, and inhibitory characteristics of XO-inhibiting peptides derived from MRJPs is still lacking. Therefore, using royal jelly as a raw material to obtain peptides with XO inhibitory activity shows promise. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a royal jelly-derived XO inhibitory peptide, its acquisition method, and its application. The screened inhibitory peptide can effectively inhibit XO activity, providing a new approach for developing xanthine oxidase inhibitory products.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A royal jelly-derived XO inhibitory peptide, wherein the amino acid sequence of the XO inhibitory peptide is selected from one or more of FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR, and YYVNTEQFR.

[0007] The method for obtaining royal jelly-derived XO inhibitory peptides includes the following steps: S1. Raw material pretreatment: Royal jelly is used as raw material. The main protein of royal jelly is extracted and a royal jelly main protein solution is prepared. Then, the royal jelly main protein solution is pretreated by high hydrostatic pressure to obtain a pretreated main protein solution. S2. Enzymatic hydrolysis: The pretreated main protein solution was continuously hydrolyzed with pepsin and trypsin, followed by enzyme inactivation and centrifugation. The supernatant was filtered and freeze-dried to obtain the main protein hydrolysis product. S3. Peptidomics identification: Liquid chromatography-tandem mass spectrometry was used to identify peptides in the above-mentioned major proteolytic products, and to obtain information on peptide molecular weight, amino acid sequence, source protein, identification confidence level and relative abundance. S4. Candidate peptide screening and identification: Peptides with high confidence in mass spectrometry identification, detectable relative abundance in enzymatic digestion products, suitable peptide length for chemical synthesis and in vitro verification, containing aromatic, hydrophobic, acidic or basic amino acid residues in the sequence, good water solubility and synthetic feasibility, and traceable origin to the main protein of royal jelly were screened. Six candidate peptides were identified: FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR and YYVNTEQFR.

[0008] Preferably, in step S1, the high hydrostatic pressure pretreatment pressure is 50-600 MPa, the pressure holding time is 5-20 min, the initial temperature is 20℃, and the pressure increase rate is 300 MPa / min.

[0009] Preferably, in step S2, the method of continuous enzymatic hydrolysis using pepsin and trypsin involves first adjusting the pH of the pretreated main protein solution to 2.0, adding pepsin at a pepsin-to-substrate mass ratio of 1:100, and hydrolyzing at 37°C for 2 hours; then adjusting the system to pH 6.9, adding trypsin at a trypsin-to-substrate mass ratio of 1:50, and continuing enzymatic hydrolysis at 37°C for 4 hours.

[0010] An XO-inhibiting composition comprising an XO-inhibiting peptide that is any one or more of the amino acids FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR, and YYVNTEQFR, or a pharmaceutically acceptable salt, isotope label, or a conjugate thereof of the above XO-inhibiting peptides with a carrier, or at least one thereof.

[0011] Preferably, the carrier is a pharmaceutically acceptable carrier, excipient, stabilizer, encapsulating material, or a combination thereof.

[0012] Preferably, the dosage form of the composition is any one of lyophilized powder, solution, powder, granules, tablets or capsules.

[0013] The above composition is used to prepare xanthine oxidase inhibitors.

[0014] This invention provides a royal jelly-derived XO inhibitory peptide, its acquisition method, and its application. Compared with existing technologies, its advantages are as follows: (1) The six MRJPs-derived candidate peptides with clearly defined sequences, namely FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR and YYVNTEQFR, obtained by this invention have been verified to have XO inhibitory activity through artificial synthesis and in vitro experiments, and have high application value.

[0015] (2) The present invention uses high hydrostatic pressure pretreatment combined with continuous enzymatic hydrolysis by pepsin and trypsin, which can regulate the conformation and aggregation state of MRJPs, improve the accessibility of some enzyme cleavage sites, and promote the release of short peptides. It was also determined that the enzymatic hydrolysis products of MRJPs obtained by 200 MPa treatment showed strong XO inhibitory activity, which provided experimental basis for the selection of high hydrostatic pressure treatment conditions.

[0016] (3) This invention verified that the IC50 of FDYDPK is 2.59 mg / mL and exhibits mixed inhibition, indicating that it can affect the substrate binding and catalytic process of XO. Furthermore, this invention establishes a complete technical route from pretreatment of royal jelly main proteins, continuous enzymatic hydrolysis, peptide identification to activity verification, which can provide a technical basis for the preparation and application of naturally derived XO inhibitory peptides. Attached Figure Description

[0017] Figure 1 This is a schematic diagram showing the XO inhibition rate of the main proteolytic products of royal jelly under different high hydrostatic pressure treatment conditions in this embodiment of the invention. Figure 2 This is a schematic diagram of the XO inhibition rate of the candidate peptide FDYDPK in the embodiments of the present invention; Figure 3 This is a schematic diagram of Lineweaver-Burk double reciprocal analysis of the candidate peptide FDYDPK inhibiting XO in an embodiment of the present invention; Figure 4 This is a schematic diagram of the XO inhibition rate of the candidate peptide LSHL in the embodiments of the present invention; Figure 5 This is a schematic diagram of Lineweaver-Burk double reciprocal analysis of the candidate peptide LSHL inhibiting XO in an embodiment of the present invention; Figure 6 This is a schematic diagram of the XO inhibition rate of the candidate peptide LTSNTFDYDPK in the embodiments of the present invention; Figure 7 This is a schematic diagram of Lineweaver-Burk double reciprocal analysis of the candidate peptide LTSNTFDYDPK inhibiting XO in an embodiment of the present invention; Figure 8This is a schematic diagram of the XO inhibition rate of the candidate peptide NYPFDVDR in the embodiments of the present invention; Figure 9 This is a schematic diagram of Lineweaver-Burk double reciprocal analysis of the candidate peptide NYPFDVDR inhibiting XO in an embodiment of the present invention; Figure 10 This is a schematic diagram of the XO inhibition rate of the candidate peptide YVNTEQFR in the embodiments of the present invention; Figure 11 This is a schematic diagram of Lineweaver-Burk double reciprocal analysis of the candidate peptide YVNTEQFR inhibiting XO in an embodiment of the present invention; Figure 12 This is a schematic diagram of the XO inhibition rate of candidate peptide YYVNTEQFR in an embodiment of the present invention; Figure 13 This is a schematic diagram of the XO inhibition rate of the positive control peptide LWM in the embodiments of the present invention; Figure 14 This is a schematic diagram of Lineweaver-Burk double reciprocal analysis of the positive control peptide LWM inhibiting XO in an embodiment of the present invention; Figure 15 IC50 of different candidate peptides inhibiting XO activity in the embodiments of the present invention 50 Value diagram; Figure 16 A schematic diagram of the molecular docking results between the candidate peptide FDYDPK and XO in an embodiment of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1: Extraction of XO inhibitory peptides: 1. Extraction and high hydrostatic pressure treatment of main proteins in royal jelly Weigh 40 g of fresh royal jelly and add 400 mL of ultrapure water. Mix thoroughly and stir magnetically at 600 r / min for 1 h under ice bath conditions. Centrifuge the mixture at 12,000 r / min for 15 min, collect the supernatant, and filter it through a 0.45 μm microporous membrane to obtain the crude MRJPs extract.

[0020] Protein concentration was determined spectrophotometrically, and the concentration was adjusted to 12 mg / mL using ultrapure water. The pH of the MRJPs solution was adjusted to 4.0 using 1 mol / L HCl or 1 mol / L NaOH.

[0021] The MRJPs solution was sealed and placed in a high hydrostatic pressure apparatus, using water as the pressure transmission medium. The initial temperature was set to 20 °C, and the pressurization rate was set to 300 MPa / min. Pressure levels of 50, 100, 200, 400, and 600 MPa were set, with holding times of 5, 10, 15, and 20 min, respectively. The MRJPs solution without high hydrostatic treatment served as a control. Immediately after treatment, the samples were stored at 4 °C.

[0022] 2. Continuous enzymatic hydrolysis of pepsin and trypsin and preparation of hydrolysis products Take 40 mL of each of the above groups of samples, adjust the pH of the system to 2.0 using 1 mol / L HCl and 1 mol / L NaOH, add pepsin at a pepsin to substrate mass ratio of 1:100, and stir at 37℃ for 2 h for enzymatic hydrolysis.

[0023] After pepsin hydrolysis, the pH of the system was adjusted to 6.9, and trypsin was added at a mass ratio of 1:50 to substrate. Hydrolysis was continued at 37°C with stirring for 4 h. The pH was monitored during hydrolysis, and maintained at approximately 6.9 using 1 mol / L HCl and 1 mol / L NaOH.

[0024] After enzymatic hydrolysis, the pH of the system was adjusted to 7.0, and the enzyme was inactivated in a boiling water bath for 10 min. After the sample was cooled to room temperature, it was centrifuged at 4℃ and 12,000 r / min for 20 min. The supernatant was collected, filtered through a 0.45 μm filter membrane, and freeze-dried to obtain the MRJPs enzymatic hydrolysis product.

[0025] The XO inhibitory activity of the enzymatic hydrolysates (hydrolysates) of each group of MRJPs was tested: The reaction system was prepared using 75 mmol / L phosphate buffer. 50 μL of enzyme digest, 50 μL of 2 mmol / L xanthine solution, and 50 μL of phosphate buffer were mixed and pre-incubated at 37°C in the dark for 15 min; then 50 μL of XO working solution (0.02 U / mL) was added to initiate the reaction. After incubation at 3°C ​​for 15 min, the absorbance at 292 nm was measured.

[0026] The concentration gradients of the enzyme hydrolysate solutions were set to 1.25, 2.5, 5, and 10 mg / mL. The XO inhibition rate was calculated using the following formula: XO inhibition rate (%) = [1 - (As - Ab) / (Ac - A0)] × 100%. Wherein, As is the absorbance of the sample reaction group, Ab is the absorbance of the sample blank group, Ac is the absorbance of the enzyme reaction group, and A0 is the absorbance of the enzyme blank group.

[0027] Curves were plotted based on the inhibition rates of enzymatic hydrolysates obtained under different high hydrostatic pressure treatment conditions and enzymatic hydrolysates of different concentrations. Specific results can be found in [the table below]. Figure 1 Under the tested conditions, the enzymatic hydrolysate obtained by treatment at 200 MPa showed strong XO inhibition activity. Subsequently, the enzymatic hydrolysate obtained under high hydrostatic pressure of 200 MPa was used for experiments.

[0028] 3. Peptidomics identification Peptidomics analysis of the MRJPs enzymatic hydrolysis products prepared under the above 200 MPa conditions was performed using ultra-high performance liquid chromatography-high resolution tandem mass spectrometry. The MRJPs enzymatic hydrolysate was reduced with dithiothreitol (DTT) to a final concentration of 10 mmol / L in a 56°C water bath for 1 h to achieve a final concentration of iodoacetamide (IAM) of 20 mmol / L. Alkylation was then performed by reacting at room temperature in the dark for 40 min. Desalting and purification were then carried out using a C18 StageTip. The desalting column was activated twice with 300 μL of 100% acetonitrile (ACN), equilibrated twice with 200 μL of an aqueous solution containing 0.1% trifluoroacetic acid (TFA), loaded twice, desalted twice with 200 μL of 0.1% TFA aqueous solution, and finally eluted once with a mixed solution of 300 μL containing 80% acetonitrile and 20% 0.1% trifluoroacetic acid. After vacuum drying, the solution was used for instrumental analysis.

[0029] Peptides were separated using an Easy-nLC 1200 nano-scale liquid chromatography system. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and 0.1% formic acid-80% acetonitrile (B). After gradient elution, the samples were injected into a Q Exactive Orbitrap mass spectrometer. Data were acquired in DDA mode (MS1 resolution 70,000, MS2 resolution 17,500, TopN=20, NCE=28). The raw mass spectrometry data were identified using PEAKSStudio software based on the UniProt database. The fixed modification was cysteine ​​alkylation, and the variable modifications included methionine oxidation and N-terminal acetylation. The mass errors were set at 20 ppm (MS1) and 0.02 Da (MS2) to achieve high-confidence peptide identification and analysis.

[0030] 4. Candidate peptide screening and identification: Based on the peptidomics identification results obtained above, candidate peptides were screened. The following factors were considered during the screening: high confidence level of mass spectrometry identification of peptides; detectable relative abundance in enzymatic digests; peptide length suitable for chemical synthesis and in vitro verification; presence of aromatic, hydrophobic, acidic, or basic amino acid residues in the sequence; good water solubility and synthetic feasibility; and traceability to the main proteins of royal jelly.

[0031] Six candidate peptides were identified through screening: FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR, and YYVNTEQFR. The sequence information of the candidate peptides is shown in Table 1. Table 1 5. Synthesis and Characterization of Candidate Peptides The candidate peptides screened above were synthesized using the Fmoc solid-phase peptide synthesis method. After synthesis, the peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the molecular weight of the target peptides was confirmed by mass spectrometry (MS). The purity of each synthesized peptide was determined by HPLC, and the purity was not less than 98%.

[0032] Before performing in vitro activity verification on the candidate peptide, the candidate peptide is dissolved in a suitable solvent and prepared into working solutions of different concentrations.

[0033] 6. Determination of XO inhibitory activity of candidate peptides The reaction system was prepared using 75 mmol / L phosphate buffer. 50 μL of candidate peptide solution, 50 μL of 2 mmol / L xanthine solution, and 50 μL of phosphate buffer were mixed and pre-incubated at 37 °C in the dark for 15 min; then 50 μL of XO working solution (0.02 U / mL) was added to initiate the reaction. After incubation at 37 °C for 15 min, the absorbance at 292 nm was measured.

[0034] Candidate peptides were used in concentration gradients of 0.1, 1, 5, and 10 mg / mL. Systems without candidate peptides served as enzyme control, and corresponding sample and enzyme blanks were included; LWM was used as the positive control peptide. Each treatment was performed in triplicate.

[0035] XO inhibition rate is calculated using the following formula: XO inhibition rate (%) = [1-(As-Ab) / (Ac-A0)]×100%.

[0036] Where As is the absorbance of the sample reaction group, Ab is the absorbance of the sample blank group, Ac is the absorbance of the enzyme reaction group, and A0 is the absorbance of the enzyme blank group.

[0037] Dose-response curves were plotted based on the inhibition rates of candidate peptides at different concentrations, and IC50 was calculated using nonlinear regression. 50The results showed that each candidate peptide exhibited varying degrees of concentration-dependent XO inhibition. Among them, FDYDPK showed the highest IC50 concentration. 50 The concentration was 2.59 mg / mL, and its inhibitory activity was on the same order of magnitude as the positive control peptide LWM; LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR, and YYVNTEQFR also showed detectable XO inhibitory activity. Related results can be found in [link to relevant results]. Figure 2 , Figure 4 , Figure 6 , Figure 8 , Figure 10 , Figure 12 , Figure 13 and Figure 15 7. XO enzyme kinetic analysis The XO inhibition type of candidate peptides was analyzed using the Lineweaver-Burk double reciprocal method. The initial reaction rate was determined under different candidate peptide concentrations and different xanthine substrate concentrations, and a double reciprocal curve was plotted with 1 / V as the ordinate and 1 / [S] as the abscissa.

[0038] As FDYDPK concentration increased, both the slope and y-intercept of the fitted line changed, indicating that apparent Vmax and Km were simultaneously affected, thus FDYDPK exhibited mixed inhibition. Double reciprocal analyses of LSHL, LTSNTFDYDPK, NYPFDVDR, and YVNTEQFR also showed varying degrees of mixed inhibition characteristics. Related results can be found in... Figure 3 , Figure 5 , Figure 7 , Figure 9 and Figure 11 .

[0039] 8. Molecular docking of FDYDPK and XO The three-dimensional structure of FDYDPK was constructed using Avogadro technology, and its geometry was optimized under the MMFF94s force field. The XO crystal structure was selected using PDB ID: 2E1Q. After removing water molecules and ligands irrelevant to the study, the receptor protein and peptide were subjected to hydrogenation, charge treatment, and structural preparation, respectively.

[0040] The docking box parameters were set to center_x=31.1, center_y=17.7, center_z=172.9, size_x=25.1, size_y=28.4, size_z=65.2. Molecular docking was performed using AutoDock Vina, and the docking conformation was visualized and analyzed using PyMOL and Discovery Studio.

[0041] The results show that FDYDPK can enter the potential binding region of XO and form hydrogen bonds, electrostatic interactions, and hydrophobic interactions with multiple amino acid residues. The optimal docking conformation has a binding energy of -8.6 kcal / mol. These results suggest, at the structural level, the potential for FDYDPK to bind with XO. (See related results in...) Figure 16 .

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A royal jelly-derived XO inhibitory peptide, characterized in that, The amino acid sequence of the XO inhibitory peptide is selected from one or more of FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR, and YYVNTEQFR.

2. A method for obtaining royal jelly-derived XO inhibitory peptide as described in claim 1, characterized in that, The acquisition method includes the following steps: S1. Raw material pretreatment: Royal jelly is used as raw material. The main protein of royal jelly is extracted and a royal jelly main protein solution is prepared. Then, the royal jelly main protein solution is pretreated by high hydrostatic pressure to obtain a pretreated main protein solution. S2. Enzymatic hydrolysis: The pretreated main protein solution was continuously hydrolyzed with pepsin and trypsin, followed by enzyme inactivation and centrifugation. The supernatant was filtered and freeze-dried to obtain the main protein hydrolysis product. S3. Peptidomics identification: Liquid chromatography-tandem mass spectrometry was used to identify peptides in the above-mentioned major proteolytic products, and to obtain information on peptide molecular weight, amino acid sequence, source protein, identification confidence level and relative abundance. S4. Candidate peptide screening and identification: Peptides with high confidence in mass spectrometry identification, detectable relative abundance in enzymatic digestion products, suitable peptide length for chemical synthesis and in vitro verification, containing aromatic, hydrophobic, acidic or basic amino acid residues in the sequence, good water solubility and synthetic feasibility, and traceable origin to the main protein of royal jelly were screened. Six candidate peptides were identified: FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR and YYVNTEQFR.

3. The acquisition method according to claim 2, characterized in that: In step S1, the high hydrostatic pressure pretreatment pressure is 200 MPa, the pressure holding time is 5-20 min, the initial temperature is 20℃, and the pressure increase rate is 300 MPa / min.

4. The acquisition method according to claim 2, characterized in that: In step S2, the method of continuous enzymatic hydrolysis using pepsin and trypsin involves first adjusting the pH of the pretreated main protein solution to 2.0, adding pepsin at a pepsin-to-substrate mass ratio of 1:100, and hydrolyzing at 37°C for 2 h; then adjusting the system to pH 6.9, adding trypsin at a trypsin-to-substrate mass ratio of 1:50, and continuing enzymatic hydrolysis at 37°C for 4 h.

5. An XO-inhibiting composition, characterized in that, The composition comprises an XO-inhibiting peptide that is any one or more of the amino acids FDYDPK, LSHL, LTSNTFDYDPK, NYPFDVDR, YVNTEQFR, and YYVNTEQFR, or a pharmaceutically acceptable salt, isotope label, or a conjugate thereof of the above XO-inhibiting peptides.

6. The composition according to claim 5, characterized in that: The carrier is a pharmaceutically acceptable carrier, excipient, stabilizer, encapsulating material, or a combination thereof.

7. The composition according to claim 5, characterized in that: The dosage form of the composition is any one of lyophilized powder, solution, powder, granules, tablets or capsules.

8. Use of the composition according to any one of claims 5-7 in the preparation of xanthine oxidase inhibitors.

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