Camel milk polypeptide with xanthine oxidase inhibitory activity and application thereof

Abstract

The invention discloses camel milk polypeptide with xanthine oxidase inhibitory activity and application thereof.The camel milk polypeptide is prepared by taking fresh camel milk as a raw material, conducting low-temperature centrifugation to remove fat, conducting triple enzymolysis through papain, pepsin and trypsin to obtain enzymatic hydrolysate, enabling the enzymatic hydrolysate to pass through an ultrafiltration membrane (the molecular weight cutoff is 10 kDa) to obtain filtrate, and conducting freeze-drying to obtain the camel milk polypeptide. The compound has xanthine oxidase inhibitory activity. The invention also discloses a small molecule polypeptide with xanthine oxidase inhibitory activity. The filtered solution is screened and combined with polypeptides by adopting a xanthine oxidase affinity ultrafiltration method, the activity of the polypeptides is verified through molecular docking and in-vitro enzyme inhibition, four small-molecule polypeptides with xanthine oxidase inhibitory activity are obtained, the sequence of the small-molecule polypeptides is shown as any one of SEQ ID NO.1-4, and the small-molecule polypeptides can be used for preparing drugs with the effect of inhibiting the uric acid level.

Description

Technical Field

[0001] This invention belongs to the technical field of enzymatic hydrolysis processing products, and relates to four camel milk polypeptides with xanthine oxidase inhibitory activity and their applications. Background Technology

[0002] Camel milk is a nutrient-rich natural dairy product, abundant in protein, whey protein, immunoglobulins, vitamins, and minerals. Its protein amino acid composition closely resembles the essential amino acid pattern for humans, exhibiting high bioavailability and nutritional value. Furthermore, its various active ingredients endow it with antibacterial, anti-disease, and immunomodulatory activities, demonstrating promising potential for functional food development. However, existing studies have shown that camel milk itself does not have a significant uric acid-lowering effect, limiting its direct application in hyperuricemia-related diseases. Therefore, further releasing its potential functional activities through enzymatic hydrolysis is crucial. Enzymatic hydrolysis utilizes proteases to directionally degrade proteins into active peptides with specific sequences under suitable conditions. This method offers advantages such as mildness, high controllability, and preservation of functional structures. Commonly used enzymes include pepsin, flavor proteases, trypsin, and papain, and are widely applied in the preparation of functional peptides in the food and pharmaceutical fields.

[0003] Affinity ultrafiltration-mass spectrometry (AUS-MS) is a highly efficient method for screening bioactive components. This technique involves co-incubating the target protein with a complex sample (such as a mixture of peptides or natural extracts), allowing the active component with specific binding ability to bind to the target. Unbound substances are then separated by ultrafiltration, and the conjugates are eluted and their structure analyzed by mass spectrometry, thereby rapidly screening for potential functional molecules.

[0004] Hyperuricemia is a common metabolic disease characterized by abnormally high levels of uric acid in the blood, primarily caused by increased uric acid production or decreased uric acid excretion during purine metabolism catalyzed by xanthine oxidase. Uric acid accumulation is not only a direct trigger for gout attacks but is also closely related to the development of various diseases such as chronic kidney disease, hypertension, and cardiovascular disease. Currently used uric acid-lowering drugs (such as allopurinol) mainly reduce uric acid levels by inhibiting XOD (xanthine oxidase) activity. While they have good efficacy, they are accompanied by significant toxic side effects, including allergies and liver and kidney damage. Therefore, screening and developing naturally derived, highly effective, and low-toxicity XOD inhibitors has important application value in the prevention and treatment of hyperuricemia and related complications. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a camel milk polypeptide with purine oxidase inhibitory activity.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] (1) Take fresh camel milk, centrifuge at 8000g and 10℃ for 30min, remove it, remove the upper fat and lower precipitate, and separate camel milk whey protein.

[0008] (2) The concentration of camel milk whey protein was determined by BCA method, the pH was adjusted to 6-8, and protease was added at an enzyme dosage of 6000-10000 U / g. The reaction was carried out at 60-70℃ for 2-4 hours with shaking to obtain the enzymatic hydrolysate. The protease was papain, flavor protease or bromelain.

[0009] (3) Take the above enzymatic hydrolysate, adjust the pH to 2-3, add pepsin at an enzyme dosage of 2000-2500 U / ml, and react with shaking at 37℃ for 2-4 hours. Adjust the pH to 6-7 to terminate the reaction, add trypsin at an enzyme dosage of 50-100 U / ml, and continue to react with shaking at 37℃ for 2-4 hours. Finally, terminate the entire digestion process by heating at 95℃ for 10 minutes. Centrifuge the obtained product at 4℃ and 10000 r / min for 15 minutes, collect the supernatant, pass the supernatant through an ultrafiltration membrane (molecular weight cutoff of 10 kDa), and freeze-dry the filtrate to obtain camel milk polypeptide in vivo digestion material. Store at -80℃ for later use.

[0010] Preferably, in step (2), the pH is adjusted to 7.

[0011] Preferably, in step (2), the amount of enzyme added is 8000 U / g.

[0012] Preferably, in step (2), the enzymatic hydrolysis temperature is 65°C.

[0013] Preferably, in step (2), the reaction time is 3 hours.

[0014] Preferably, in step (2), the protease is papain.

[0015] Preferably, in step (3), the initial pH is 3.

[0016] Preferably, in step (3), the amount of pepsin added is 2000 U / ml.

[0017] Preferably, in step (3), the initial reaction time is 3 hours.

[0018] Preferably, in step (3), the pH of the second reaction step is 6.5.

[0019] Preferably, in step (3), the amount of trypsin added is 100 U / ml.

[0020] Preferably, in step (3), the reaction time in the second step is 3 hours.

[0021] (4) The lyophilized powder was subjected to XOD affinity ultrafiltration mass spectrometry. Peptides bound to XOD could not pass through the ultrafiltration membrane, but unbound peptides would pass through the membrane under unstable conditions. The reaction mixture consisted of 200 μL 0.2 U / mL XOD, 100 μL pH 7.4 sodium phosphate buffer, and 100 μL 10 mg / mL allopurinol. The mixture was incubated in a 37°C water bath for 30 min. Then, 200 μL of PBS containing 2 mg / mL sample was added at 37°C, and the mixture was incubated for another 30 min. Subsequently, the reaction solution was passed through a 10 kDa ultrafiltration tube. Then, 200 μL of phosphate buffer was added to wash the filtrate, and the unbound substances were washed three times. The bound peptides were eluted from XOD by adding 200 μL of 70% (v / v) methanol solution, and the solution was centrifuged at 5000 g for 5 min. Finally, the eluted solution was concentrated by centrifugation. By comparing the differences in mass spectrometry peak areas, peptides were selected for molecular docking based on the differences in peak areas.

[0022] The small molecule peptide has the amino acid sequence SEQ ID NO.1: IPVPQ.

[0023] The small molecule peptide has the amino acid sequence SEQ ID NO.2: SYDTPEGIA.

[0024] The small molecule peptide has the amino acid sequence SEQ ID NO.3: PNIDPPTVE.

[0025] The small molecule peptide has the amino acid sequence SEQ ID NO.4: NFIPPI.

[0026] (6) The structure of XOD was verified by mass spectrometry-identified peptides using Discovery Studio 3.0 software. The spatial structure of the peptides was plotted using Chem3D Ultra 8.0, and the crystal structure of the XOD enzyme (from cow's milk, PDB ID1FIQ) was obtained from the RCSB protein database. The 3D structure of the active peptide was constructed using Chem3D Ultra 8.0, and docking parameters and ligand scores were calculated to finally form a ligand-protein interaction diagram.

[0027] (7) In this invention, camel milk was treated with a combination of various enzymatic hydrolysis and affinity ultrafiltration chromatography. The enzymatic hydrolysis products and peptides such as PNIDPPTVE (IC50 of 5.365 μmol / L) with high inhibitory activity against xanthine oxidase were obtained by LC-MS / MS and in vitro enzyme inhibition identification.

[0028] The advantages of this invention over existing technologies are as follows: Through comparative experiments, this invention has found that camel milk polypeptides with XOD inhibitory activity can be extracted from fresh camel milk through triple enzymatic hydrolysis with papain, pepsin, and trypsin. In contrast, camel milk without enzymatic hydrolysis, or after enzymatic hydrolysis with only papain, papain + pepsin, or papain + trypsin, did not show the ability to inhibit XOD activity, demonstrating the rigor and superiority of the method of this invention. Furthermore, by using xanthine oxidase affinity ultrafiltration to screen for binding polypeptides, and verifying the polypeptide activity through molecular docking and in vitro enzyme inhibition, four small molecule polypeptides with xanthine oxidase inhibitory activity were obtained, which show potential application value as functional food components for regulating blood uric acid levels. Attached Figure Description

[0029] Figure 1 A comparison chart of XOD inhibition rates of camel milk protein and various proteases hydrolyzed polypeptides according to the present invention;

[0030] Figure 2 A comparison of the XOD inhibition rates of three proteases in camel milk hydrolysis and different molecular weight polypeptides according to the present invention.

[0031] Figure 3 This is a column chromatography elution diagram of enzymatic hydrolysis of ultrafiltrate according to the present invention;

[0032] Figure 4 The images show the elution profiles of peptide column chromatography using affinity ultrafiltration according to the present invention; wherein (A) is the result image of the group without positive control agent, and (B) is the result image of the group with positive control agent.

[0033] Figure 5 The images are mass spectra of affinity ultrafiltration implemented according to the present invention; wherein, (A) is the result image of the group without positive drug, and (B) is the result image of the group with positive drug;

[0034] Figure 6 This is a secondary mass spectrum of the active polypeptide according to the present invention;

[0035] Figure 7 The conformation of the camel milk polypeptide according to the present invention in the XOD pocket is shown below; wherein, (A) is the conformation diagram of IPVPQ, (B) is the conformation diagram of SYDTPEGIA, (C) is the conformation diagram of PNIDPPTVE, and (D) is the conformation diagram of NFIPPI.

[0036] Figure 8The diagram shows the binding mode and interaction plane of the camel milk polypeptide in the XOD active site according to the present invention; wherein, (A) is the plane corresponding to IPVPQ, (B) is the plane corresponding to SYDTPEGIA, (C) is the plane corresponding to PNIDPPTVE, and (D) is the plane corresponding to NFIPPI.

[0037] Figure 9 To illustrate the xanthine oxidase inhibitory activity of camel milk polypeptides according to the present invention; wherein, (A) is a statistical chart of XOD inhibition rate of IPVPQ, (B) is a statistical chart of XOD inhibition rate of SYDTPEGIA, (C) is a statistical chart of XOD inhibition rate of PNIDPPTVE, and (D) is a statistical chart of XOD inhibition rate of NFIPPI.

[0038] Figure 10 The effect of camel milk polypeptides on the viability of Hep G2 cells according to the present invention; wherein, (A) is a cell viability chart of IPVPQ, (B) is a cell viability chart of SYDTPEGIA, (C) is a cell viability chart of PNIDPPTVE, and (D) is a cell viability chart of NFIPPI.

[0039] Specific implementation steps

[0040] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps involved in the present invention do not preclude the possibility of adding other steps before, after, or between these steps; furthermore, these embodiments are merely illustrative of the present invention and do not constitute a limitation on the scope of protection. Furthermore, unless otherwise specified, the numbering of each step is only for ease of differentiation and does not limit their order or constrain the implementation of the present invention. Adjustments or modifications to the order of related steps, as long as they do not substantially change the technical content, are also considered to fall within the feasible scope of the present invention.

[0041] To provide a clearer understanding of the above technical solutions, exemplary embodiments of the present invention will now be described in further detail. Although exemplary embodiments of the present invention have been provided, it should be understood that the present invention can be implemented in various ways and should not be limited to the specific forms described herein. These embodiments are intended to help to more fully understand the principles of the present invention and to enable those skilled in the art to accurately grasp the overall scope of the invention.

[0042] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0043] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0044] Camel milk was produced by Xinjiang Xintuo Milk Industry; papain, pepsin, and trypsin were products of Beijing Solarbio Science & Technology Co., Ltd., with catalog numbers G8430, P8160, and T8150, respectively; xanthine oxidase was a product of Shanghai Maclean Biochemical Technology Co., Ltd., with catalog number X902417-100U; and Hep G2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHU72.

[0045] Example 1: Preparation of Camel Milk Polypeptides

[0046] Fresh camel milk from Xinjiang was placed in a 50mL centrifuge tube and centrifuged at 8000g and 10℃ for 30min. The tube was then removed, the upper fat layer and the lower precipitate were removed, and the total protein of the camel milk was obtained.

[0047] The total protein concentration of the obtained whey was determined using a BCA protein concentration assay kit. Subsequently, the pH of the protein solution was adjusted to 7.0 using dilute hydrochloric acid or sodium hydroxide solution, and papain was added at a dosage of 8000 U / g. Enzymatic hydrolysis was carried out at 65°C for 3 hours. After hydrolysis, the reaction system was heated to 95°C and held for 10 minutes to terminate enzyme activity. After the system was rapidly cooled to room temperature, the pH was adjusted to 7.0 again using dilute hydrochloric acid or sodium hydroxide. The mixture was then centrifuged at 5000 rpm at 4°C for 5 minutes, and the supernatant was collected and lyophilized to obtain camel milk peptides.

[0048] Example 2: Preparation of Simulated Digested Peptides

[0049] 5g of camel milk polypeptide was dissolved in 50mL of distilled water, and the pH was adjusted to 3. Pepsin was added at a concentration of 2000U / ml, and the mixture was reacted with shaking at 37℃ for 3 hours. The reaction was terminated by adjusting the pH to 6.5, and trypsin was added at a concentration of 100U / ml. The reaction was continued at 37℃ with shaking for another 3 hours. Finally, the entire digestion process was terminated by heating at 95℃ for 10 minutes. The resulting digestion product was centrifuged at 4℃ and 10000 rpm for 15 minutes, and the supernatant was collected to obtain the camel milk polypeptide digest. The supernatant was passed through an ultrafiltration membrane (molecular weight cutoff of 10kDa), and the filtrate was lyophilized and stored at -80℃ for later use.

[0050] Example 3 XOD Inhibition Activity

[0051] In vitro XOD inhibition experiments were performed on the prepared peptides and camel milk: peptide solutions of different concentrations were prepared with PBS, and the reaction was performed using a 96-well plate system. 10 μL of the sample solution at different concentrations and 10 μL of xanthine oxidase (XOD, 0.2 U / mL) were added to each well, followed by 180 μL of phosphate-buffered saline (PBS, pH 7.4). After pre-incubating the reaction system at 37°C for 15 min, 50 μL of xanthine solution (final concentration 0.15 mM) was added to initiate the enzymatic reaction, and incubation was continued at 37°C for another 15 min. The absorbance of each well was measured at 292 nm using a microplate reader, and the inhibition rate was calculated according to the following formula.

[0052]

[0053] In the formula, A1 is the absorbance measured with 10 μL XOD, 190 μL PBS, and 50 μL xanthine solution (positive group); A2 is the absorbance measured with 200 μL PBS and 50 μL xanthine solution (blank group); A3 is the absorbance measured with 10 μL XOD, 10 μL sample solution, 180 μL PBS, and 50 μL xanthine solution (sample group); and A4 is the absorbance measured with 10 μL sample solution, 190 μL PBS, and 50 μL xanthine solution (sample background group).

[0054] like Figure 1 As shown, the total protein group of camel milk used camel milk peptides from Example 1 without enzymatic hydrolysis. The results showed that camel milk did not exhibit the ability to inhibit xanthine oxidase (XOD) activity without enzymatic hydrolysis. Even after enzymatic hydrolysis with papain, papain + pepsin, or papain + trypsin, it still did not exhibit the ability to inhibit XOD activity.

[0055] like Figure 2 As shown, after triple enzymatic hydrolysis with papain, pepsin, and trypsin, polypeptide fragments with inhibitory activity were released. Ultrafiltration separation analysis showed that polypeptides with a molecular weight greater than 10 kDa had a significantly lower inhibitory effect on XOD than polypeptides with a molecular weight less than 10 kDa. Therefore, polypeptides with a molecular weight less than 10 kDa were selected as the target for the next step of screening for active monomeric polypeptides.

[0056] Example 4 Affinity Ultrafiltration Protein Chromatography

[0057] Peptides bound to XOD cannot pass through the ultrafiltration membrane, but unbound peptides will pass through the membrane under unstable conditions. The reaction mixture consisted of 200 μL 0.2 U / mL XOD, 100 μL pH 7.4 sodium phosphate buffer, and 100 μL 10 mg / mL allopurinol. The mixture was incubated in a 37°C water bath for 30 min. Then, 200 μL of 2 mg / mL sample was added at 37°C, and incubation was continued for another 30 min. Subsequently, the reaction solution was passed through a 10-kDa ultrafiltration tube. The filtrate was then washed three times with 200 μL of phosphate buffer to remove unbound substances. Bound peptides were eluted from XOD by adding 200 μL of 70% (v / v) methanol solution, and the solution was centrifuged at 5000g for 5 min. Finally, the eluted solution was concentrated by centrifugation. The resulting sample and total peptides with a molecular weight less than 10 kDa were then filtered through Superdex. TM The peptide 10 / 300GL gel column was used with a mobile phase of 50 mM phosphate buffer + 150 mM NaCl at pH 7.0. The sample loading volume was 100 μL, the flow rate was 0.5 mL / min, and the absorbance was measured at 265 nm and plotted as a curve.

[0058] like Figure 3 As shown, the total polypeptide protein chromatography results revealed four main components, which, after affinity ultrafiltration, were as follows: Figure 4 In the group, (A) was the group without positive drug and (B) was the group with positive drug. The main binding peptide was component 3. After the addition of the competitive inhibitor allopurinol, the peak area decreased significantly, indicating that there was a binding peptide at this site. Then, component 3 was analyzed by mass spectrometry.

[0059] Example 5 Mass Spectrometry Analysis

[0060] Desalted peptides were separated on a Nano LC U3000 liquid chromatography system (Thermo Fisher Scientific) using an Acclaim PepMap nano C18 trap column (164535, Thermo Fisher) and an Acclaim PepMap 100 C18 column (164569, Thermo Fisher) at a flow rate of 300 μL / min. The linear gradient used over 120 minutes was as follows: 2% B (ACN containing 0.1% formic acid) / 98% A (0.1% formic acid) was increased to 5% B within 10 min, from 5% B to 19% B within 70 min, then from 19% B to 30% B within 15 min, from 30% B to 98% B within 10 min, returned to 2% B within 5 min, and then held at 2% B for 2 minutes. Mass spectrometry data were acquired using a Fusion Lumos Tribrid (ThermoFisherScientific) mass spectrometer in data-dependent acquisition mode, covering a full scan from m / z 300 to 1550. Precursor ions were sequentially dissociated using high-energy collisions (HCDs). The normalized collision energy was set to 30%. The dynamic exclusion time was 30 s. The automatic gain control for MS1 and MS2 was set to 1e6 and 5e4, respectively, and the resolutions for MS1 and MS2 were set to 120 K and 15 K, respectively.

[0061] Get as Figure 5 Based on the differences in peak shape, the required active peptides were identified from the mass spectra shown. The amino acid sequences of the active peptides were determined by combining the secondary mass spectra: IPVPQ, PNIDPPTVE, SYDTPEGIA, and NFIPPI.

[0062] Example 6 Molecular docking analysis

[0063] The activity of peptides identified by mass spectrometry was verified using Discovery Studio 3.0 software based on molecular docking. The spatial structure of the peptides was plotted using Chem3D Ultra 8.0, and the crystal structure of the XOD enzyme (from cow's milk, PDB ID1FIQ) was obtained from the RCSB protein database. The peptide structure was minimized using the CHARMm simulation program in Discovery Studio. After removing water molecules and adding hydrogen atoms to the receptor target, the active site (x, yz) and docking radius (r) were defined in the "Receptor LigandInteractions" module. Molecular docking between the receptor and peptide was performed using the CDOCKER program. The results were analyzed based on the highest -CDOCKEREnergy score, combined with indicators such as free energy, hydrogen bonds, hydrophobic groups, and charged groups.

[0064] The results are shown in the figure. Figure 7 This shows the conformation of the four polypeptides in the XOD pocket. Figure 8 This is a binding plane diagram of the four polypeptides at the XOD active site.

[0065] IPVPQ interacts with Phe798 and Ser1080 residues of xanthine oxidase (XOD) via π–π stacking interactions on the benzene rings of multiple phenylalanine residues, while also forming hydrophobic interactions with Leu873 and Val1011 residues. Furthermore, this polypeptide can form a multi-layered stable hydrogen bond network with residues such as Arg880, Arg912, Gly1039, Gly1193, Gln1194, and Glu1261, effectively hindering the entry of purine substrates into the enzyme's active site, thereby inhibiting the catalytic activity of xanthine oxidase.

[0066] NFIPPI binds stably to the active site of xanthine oxidase (XOD) through multiple binding mechanisms. It significantly enhances the binding affinity of the complex by forming a multiple hydrogen bond network with residues such as Ser347, Thr262, Gly350, and Glu263, and by forming stable salt bridge interactions with Arg428 and Glu263. Furthermore, the hydrophobic groups of NFIPPI form hydrophobic associations and π-alkyl interactions with residues such as Phe337, Val342, and Val345, further enhancing binding stability and effectively preventing purine substrates from entering the enzyme's active site, thereby inhibiting the catalytic activity of xanthine oxidase.

[0067] PNIDPPTVE binds stably to the active site of xanthine oxidase (XOD) through multiple mechanisms of action. It forms multiple hydrogen bonds with key residues such as Asn261, Gly260, Ser347, and Glu263, while simultaneously forming salt bridges and electrostatic attraction with Arg426 and Lys433, greatly enhancing binding stability. Furthermore, the hydrophobic residues of this peptide form van der Waals interactions with Val259, Ala346, and Phe270, effectively preventing purine substrates from entering the enzyme's active site, thereby inhibiting the catalytic activity of xanthine oxidase.

[0068] SYDTPEGIA binds stably to the active site of xanthine oxidase (XOD) through multiple binding mechanisms. It forms multiple conventional hydrogen bonds with key residues such as Thr25, Gly44, Glu676, and Arg32, while simultaneously forming electrostatic attraction with residues such as Arg598 and Asp594, significantly enhancing binding stability. Furthermore, this peptide also forms van der Waals interactions with residues such as Met268, Leu41, and Ala678, effectively preventing purine substrates from entering the enzyme's active site, thereby inhibiting the catalytic activity of xanthine oxidase.

[0069] Example 7 Synthesis of Polypeptides

[0070] The peptides were synthesized using a solid-phase synthesis method. Peptide purity was determined by HPLC. The column types were Inertsil ODS-3 4.6 x 250 mm 5 μm and Inertsil ODS-SP 4.6 x 250 mm 5 μm. Mobile phase A was trifluoroacetic acid / water = 0.065% / 100% (v / v), and mobile phase B was trifluoroacetic acid / acetonitrile = 0.05% / 100% (v / v). The flow rate was 1 ml / min, the column temperature was 35℃, and the detection wavelength was 220 nm. Within 25 min, mobile phase B increased from 5% to 65%.

[0071] Example 8: XOD inhibitory activity of monomeric peptides

[0072] The polypeptide was prepared to the appropriate concentration with PBS, and the in vitro XOD inhibition rate of the synthesized polypeptide was determined according to the method in Example 2.

[0073] The results are as follows Figure 9 As shown, IPVPQ, PNIDPPTVE, SYDTPEGIA, and NFIPPI all exhibited some inhibitory activity against xanthine oxidase. PNIDPPTVE showed the best inhibitory activity, with an IC50 of 5.365 μmol / L. The IC50s for IPVPQ and SYDTPEGIA were 6.054 μmol / L and 11.996 μmol / L, respectively. Furthermore, the inhibitory effect of NFIPPI on XOD plateaued after reaching a certain concentration within the experimentally defined range.

[0074] Table 1. Monomeric peptide information and XOD inhibition rate.

[0075] polypeptide sequence molecular weight Docking score GRAVY <![CDATA[IC 50 (μmol / L)]]> IPVPQ 552.66 g / mol -9.07664 0.4 6.054 PNIDPPTVE 981.05 g / mol -7.8259 -0.81 5.365 SYDTPEGIA 951.97 g / mol -8.44702 -0.61 11.996 NFIPPI 699.83 g / mol -9.8847 0.85

[0076] Example 9: Effect of monomeric peptides on Hep G2 cell viability

[0077] Normal Hep G2 cells were cultured in DMEM complete medium. Healthy Hep G2 cells were then cultured at a density of 1 x 10-1 cells per well. 4 Cells were seeded at a density of 100 μL in 96-well plates and pre-incubated overnight at 37°C with 5% CO2. The plates were divided into a control group and an experimental group. The control group was given DMEM complete medium, while the experimental group was given different concentrations of camel milk peptide (1.25 μM, 2.5 μM, 5.0 μM, 10.0 μM, 20.0 μM, and 40.0 μM), 100 μL per well. After 24 h of intervention, 10 μL of LCK-8 assay solution and 100 mL of serum-free DMEM medium were added to each well, and the plates were incubated at 37°C with 5% CO2 for 1 h. The absorbance at 450 nm was measured using a microplate reader.

[0078] The results are as follows Figure 10 As shown, after 24 hours of intervention, the IPVPQ group effectively promoted the proliferation of Hep G2 cells without cytotoxicity; PNIDPPTVE inhibited the proliferation of Hep G2 cells at 40.0 μM, showing certain cytotoxicity; SYDTPEGIA and NFIPPI both inhibited the proliferation of Hep G2 cells.

Claims

1. A camel milk polypeptide, characterized in that, Its amino acid sequence is shown in any one of SEQ ID NO.1-4.

2. A method for preparing a camel milk polypeptide with xanthine oxidase inhibitory activity, characterized in that, Includes the following steps: Camel milk whey protein was separated from camel milk by centrifugation. The pH was adjusted to 6-8. Based on the camel milk whey protein content, protease was added at a dosage of 6000-10000 U / g, and the mixture was reacted with shaking at 60-70℃ for 2-4 hours to obtain the enzymatic hydrolysate. The pH was adjusted to 2-3, and pepsin was added at a dosage of 2000-2500 U / ml, and the mixture was reacted with shaking at 37℃ for 2-4 hours. The reaction was terminated by adjusting the pH to 6-7. Trypsin was added at a dosage of 50-100 U / ml, and the mixture was reacted with shaking at 37℃ for 2-4 hours to terminate digestion. Centrifuge, collect the supernatant, filter, collect the filtrate, and freeze-dry.

3. The preparation method according to claim 2, characterized in that, The method of separating camel milk whey protein from camel milk by centrifugation includes: centrifuging at 8000g and 10℃ for 30min to remove the upper fat and lower precipitate to obtain camel milk whey protein.

4. The preparation method according to claim 2, characterized in that, The digestion is terminated by heating at 95°C for 10 minutes; the centrifugation, collecting the supernatant, is performed by centrifuging at 4°C and 10,000 r / min for 15 minutes; the filtration, collecting the filtrate, is performed by filtering with an ultrafiltration membrane with a molecular weight cutoff of 10 kDa.

5. The preparation method according to claim 2, characterized in that, The protease is papain, flavor protease, or bromelain.

6. Camel milk polypeptide with xanthine oxidase inhibitory activity prepared by the preparation method according to any one of claims 2-5.

7. The use of the camel milk polypeptide of claim 1 or claim 6 in the preparation of a drug having the effect of inhibiting uric acid levels.

8. The use of the camel milk polypeptide according to claim 1 or claim 6 in the preparation of purine oxidase inhibitors.

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