A whitevein peptid with xanthine oxidase inhibitory activity, and a preparation method and application thereof

Abstract

The application discloses a whitevein milkwort peptide with xanthine oxidase inhibiting activity, a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The amino acid sequence of the whitevein milkwort peptide is FNW or YGW. The application finds and proves that there is a polypeptide component with xanthine oxidase inhibiting activity in whitevein milkwort hydrolysate, wherein the whitevein milkwort peptides with the amino acid sequences of FNW and YGW can effectively inhibit the activity of xanthine oxidase, have the function of reducing the uric acid level, and can be used for preparing pharmaceutical preparations for relieving hyperuricemia.

Description

Technical Field

[0001] This invention relates to a small molecule peptide, its preparation method and application, specifically to a white peony peptide with xanthine oxidase inhibitory activity, its preparation method and application, belonging to the field of bioactive peptide technology. Background Technology

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder, mainly characterized by abnormally high levels of uric acid in the blood. Long-term accumulation of urate in the body can not only induce kidney inflammation but also lead to kidney dysfunction and damage. Hyperuricemia is closely related to gout and is also an important risk factor for many chronic diseases such as hypertension, diabetes, and atherosclerosis. Xanthine oxidase is a key enzyme in purine metabolism, a molybdenum-containing flavin enzyme complex composed of two identical subunits. It catalyzes the conversion of hypoxanthine to xanthine, and further converts xanthine to uric acid. Therefore, inhibiting the activity of xanthine oxidase has become an important strategy for regulating uric acid levels and intervening in the progression of hyperuricemia.

[0003] Currently, commonly used xanthine oxidase inhibitors in clinical practice mainly include allopurinol and febuxostat. These drugs have significant uric acid-lowering effects, but may cause varying degrees of adverse reactions during clinical use. Therefore, developing safe and effective natural drug alternatives has important clinical value.

[0004] In recent years, bioactive peptides have attracted much attention as a novel type of component in the preparation of functional foods and drug development. Among them, food-derived xanthine oxidase inhibitory peptides have become a research hotspot due to their high biocompatibility and low toxicity. Baiwei, a traditional Chinese medicinal herb, has been proven to possess various bioactivities. Current research mainly focuses on its C... 21 Research on the bioactivity of steroidal and polysaccharide components is limited, but studies on the bioactive peptides obtained from their hydrolysis are still lacking. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to: hydrolyze Cynanchum paniculatum and screen out Cynanchum paniculatum peptides with good xanthine oxidase inhibitory activity from the active peptides obtained by hydrolysis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A white peony peptide with xanthine oxidase inhibitory activity, wherein the amino acid sequence of the white peony peptide is FNW or YGW.

[0007] The aforementioned method for preparing the white peony peptide with xanthine oxidase inhibitory activity employs a solid-phase synthesis method, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, to synthesize peptides with amino acid sequences of FNW or YGW.

[0008] The aforementioned application of Baiwei peptide, which has xanthine oxidase inhibitory activity, in the preparation of drugs to alleviate hyperuricemia.

[0009] The advantages of this invention are: This invention discovers and confirms that there are polypeptide components with xanthine oxidase inhibitory activity in the hydrolysate of Cynanchum paniculatum. Among them, Cynanchum paniculatum peptide with amino acid sequences FNW and YGW can effectively inhibit the activity of xanthine oxidase and has the effect of reducing uric acid levels. It can be used to prepare drug preparations to relieve hyperuricemia. Attached Figure Description

[0010] Figure 1 This is a 3D diagram of the optimal binding conformation of the candidate active peptide FNW to the xanthine oxidase protein molecule. Figure 2 This is a 3D diagram of the optimal binding conformation of the candidate active peptide YGW to the xanthine oxidase protein molecule. Figure 3 This is a curve showing the inhibitory activity of white peony peptide FNW on xanthine oxidase; Figure 4 This is a curve showing the inhibitory activity of YGW peptide on xanthine oxidase. Detailed Implementation

[0011] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0012] I. Obtaining Baiwei polypeptide components by hydrolysis of Baiwei A method for obtaining Baiwei polypeptide components by hydrolyzing Baiwei includes the following steps: (1) Processing raw materials and preparing homogenate The dried roots and rhizomes of Cynanchum paniculatum were ground into powder and passed through an 80-mesh cell sieve. The powder was then mixed with ultrapure water at a ratio of 1g:20ml to prepare a Cynanchum paniculatum homogenate.

[0013] (2) Preparation of Baiwei polypeptide by enzymatic hydrolysis Adjust the pH of the *Cynanchum paniculatum* homogenate obtained in step (1) to 4.8, add cellulase at a ratio of 50 U / g (based on the weight of *Cynanchum paniculatum* powder), and inactivate the enzyme after enzymatic hydrolysis at 50℃ (within the range of 50-55℃) for 120 min. After the hydrolysate cools to room temperature, centrifuge at 7000 r / min for 15 min and collect the supernatant. Adjust the pH of the supernatant to 7.0, add neutral protease at a ratio of 8000 U / g (based on the weight of *Cynanchum paniculatum* powder), and inactivate the enzyme after enzymatic hydrolysis at 50℃ (within the range of 50-55℃) for 180 min. After the hydrolysate cools to room temperature, centrifuge at 5000 r / min for 10 min and collect the supernatant.

[0014] (3) Ultrafiltration separation and purification of Baiwei polypeptide The supernatant collected in step (2) was subjected to ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 5 kDa. The filtrate with a molecular weight < 5 kDa was collected to obtain the Baiwei polypeptide component.

[0015] II. Identification and Analysis of Baiwei Peptide Sequence The sequences of the previously obtained Baiwei peptide components were determined by LC-MS / MS, and the amino acid sequences of the peptides were determined by comparison analysis using the UniProt database.

[0016] III. Screening for Baiwei peptides with both good bioactivity and safety The sequences obtained from the previous identification were used for activity prediction, novelty screening, toxicity assessment, allergenicity analysis, digestibility prediction, and stability prediction.

[0017] First, the Peptide Ranker platform was used to score the bioactivity of all peptides. Peptides with a score ≥0.5 were screened, and peptides with a score <0.5 were not further screened.

[0018] Then, peptide sequences were compared using the UniProt and BIOPEP databases to exclude known active peptides and ensure the novelty of the screened peptides.

[0019] Subsequently, the ToxinPred tool was used to assess the potential toxicity and physicochemical properties of the peptides, excluding peptides that may be cytotoxic. The AlgPred 2.0 tool was used to predict the potential allergenicity of the peptides, screening for non-allergenic peptides. The PeptideCutter tool was used to predict the digestibility of the peptides, screening for peptides lacking cleavage sites of at least one enzyme (pepsin, chymotrypsin, trypsin). The PLifePred tool was used to predict the plasma half-life of the peptides, screening for peptides with a half-life >800s, which were considered to have good stability in the blood.

[0020] The above screening criteria ensure that the bioactive peptides obtained in the final screening have both good bioactivity and safety, and can be used for subsequent synthesis, validation and product development.

[0021] Based on multi-dimensional screening considering potential activity, novelty, toxicity, allergenicity, digestibility, and stability, peptides with amino acid sequences FNW and YGW were finally selected and subjected to molecular docking analysis with xanthine oxidase.

[0022] The physicochemical properties of peptides FNW and YGW are detailed in Table 1.

[0023] Table 1 Physicochemical properties of peptides FNW and YGW

[0024] IV. Molecular docking screening The crystal structure of xanthine oxidase (PDB ID: 1FIQ) was obtained from the PDB database (http: / / www.rcsb.org / ). The protein structure was preprocessed using Pymol software (removing water molecules and existing small molecule ligands), and the preprocessed xanthine oxidase protein was used as the acceptor for molecular docking.

[0025] For the previously screened candidate active peptides FNW and YGW, their initial three-dimensional structures were constructed and energy minimization was performed. The most stable spatial conformation of the peptide was obtained through conformation optimization, which served as ligands for molecular docking.

[0026] Molecular docking was performed using AutoDock Vina, and the binding sites between candidate active peptides and xanthine oxidase protein were further analyzed.

[0027] The optimal binding conformation 3D diagram of the candidate active peptide FNW to xanthine oxidase protein is shown below. Figure 1 .

[0028] The optimal binding conformation 3D diagram of the candidate active peptide YGW to xanthine oxidase protein is shown below. Figure 2 .

[0029] Depend on Figure 1 It can be seen that the binding free energy of candidate active peptide FNW to xanthine oxidase is -9.3 kcal / mol, showing a strong binding ability; candidate active peptide FNW forms 8 hydrogen bonds with LYS249, PRO253, LYS256, GLY350, THR354, ARG394 and GLU402.

[0030] Depend on Figure 2 It can be seen that the binding free energy of the candidate active peptide YGW to xanthine oxidase is -9.6 kcal / mol, showing a strong binding ability; the candidate active peptide YGW forms 6 hydrogen bonds with LEU257, VAL259, GLY260, ASN261, GLY350 and THR354.

[0031] V. In vitro inhibitory activity of Baiwei peptide FNW and Baiwei peptide YGW against xanthine oxidase Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, peptides with amino acid sequences of FNW or YGW were synthesized by solid-phase synthesis method, and were respectively denoted as Baiwei peptide FNW and Baiwei peptide YGW.

[0032] Baiwei peptide FNW and Baiwei peptide YGW were prepared into a series of concentration gradient solutions of 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 3 mg / ml and 4 mg / ml, respectively.

[0033] Four reaction systems were set up, specifically: Group A (sample blank group): The reagents added were PBS, xanthine, and xanthine oxidase in that order; Group B (blank group): The reagents added were PBS, xanthine, and PBS in that order; Group C (experimental group): The reagents added in sequence were Baiwei peptide FNW or Baiwei peptide YGW, xanthine, and xanthine oxidase; Group D (enzyme blank group): The reagents added were, in order, Baiwei peptide FNW or Baiwei peptide YGW, xanthine, and PBS.

[0034] The reaction process is as follows: Each reaction system was first incubated with 100 μl of Baiwei peptide FNW solution, Baiwei peptide YGW solution, or phosphate buffer (PBS), followed by 50 μl of 2 mM xanthine solution. The system was then incubated at 37°C in the dark for 15 min. After that, 50 μl of 0.05 U / ml xanthine oxidase solution or PBS was added, and the system was incubated at 37°C in the dark for another 15 min.

[0035] After incubation, the absorbance of each group at 295 nm was measured.

[0036] Calculate the xanthine oxidase inhibition rate (%) using the following formula:

[0037] The calculated inhibition rates of xanthine oxidase by different concentrations of Baiwei peptide FNW are shown in the figure. Figure 3 .

[0038] The calculated inhibition rates of xanthine oxidase by different concentrations of YGW (a type of peptide) are shown in the figure below. Figure 4 .

[0039] Depend on Figure 3 and Figure 4 It can be seen that the IC50 of Baiwei peptide FNW is... 50 The IC50 of Baiwei Peptide YGW was 1.973 mg / ml (4.2385 mM). 50 The concentration was 1.854 mg / ml (4.3681 mM), and both exhibited good inhibitory activity against xanthine oxidase.

[0040] In summary, both the FNW and YGW peptides obtained by screening in this invention have good xanthine oxidase inhibitory activity, both have the effect of reducing uric acid levels, and both can be used to prepare drug formulations to relieve hyperuricemia.

[0041] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. Application of a tripeptide with the amino acid sequence FNW in the preparation of drugs to alleviate hyperuricemia.

2. Application of the tripeptide with the amino acid sequence YGW in the preparation of drugs to alleviate hyperuricemia.

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