CN113004370B

CN113004370B - Dipeptide capable of relieving hyperuricemia and regulating intestinal flora and application thereof

Info

Publication number: CN113004370B
Application number: CN202110173724.6A
Authority: CN
Inventors: 卢国定; 芦晨阳
Original assignee: Ningbo Green Health Pharmaceutical Co ltd
Current assignee: Ningbo Green Health Pharmaceutical Co ltd
Priority date: 2021-02-09
Filing date: 2021-02-09
Publication date: 2022-06-17
Anticipated expiration: 2041-02-09
Also published as: CN113004370A

Abstract

The invention discloses a dipeptide for relieving hyperuricemia and regulating intestinal flora and application thereof, and is characterized in that the amino acid sequence of the dipeptide is as follows: the dipeptide has the application in the aspects of preparing xanthine oxidase inhibitors, preparing uric acid-reducing medicines or preparing gout disease adjuvant therapy medicines and intestinal flora regulators, and has the advantages of inhibiting xanthine oxidase activity, reducing uric acid activity and regulating intestinal flora.

Description

Dipeptide capable of relieving hyperuricemia and regulating intestinal flora and application thereof

Technical Field

The invention relates to the technical field of polypeptides, in particular to a dipeptide capable of relieving hyperuricemia and adjusting intestinal flora and application thereof.

Background

Hyperuricemia is a metabolic disease caused by purine metabolic disorder, and is frequently seen in coastal areas, and a high-purine diet and a high-sugar diet are important causes of hyperuricemia. Hyperuricemia is clinically manifested by abnormal rise of blood uric acid content, and increased risk of gouty arthritis and nephropathy. Clinical drugs treat hyperuricemia mainly by inhibiting uric acid synthesis (allopurinol and the like) and promoting uric acid excretion (probenecid and the like), wherein xanthine oxidase is a main target of drugs inhibiting uric acid synthesis. Clinical medicines cannot be used for daily health care, and various problems that toxic and side effects and long-term safety cannot be guaranteed exist possibly, so that the food-borne efficacy factors with the uric acid reducing activity are expected to be searched for to deal with the increasingly severe risk of hyperuricemia according to the concept of food-medicine homology. The aquatic protein oligopeptide is a mixture prepared by hydrolyzing animal and plant proteins, and has proved to have the function of relieving hyperuricemia. However, the number of core uric acid lowering efficacy molecules in the oligopeptide raw material in the form of a mixture has not been clearly resolved.

The intestinal flora is a general term for microorganisms which colonize the human intestinal tract. Recent studies have shown that the intestinal flora plays an important role in the development of hyperuricemia. It is known that the development of hyperuricemia depends on the presence of the intestinal flora, and that the modification of the intestinal flora by probiotics, accompanied by the structure and metabolism of the intestinal flora, can alleviate hyperuricemia, while the uric acid lowering activity of active peptides is partially mediated by the intestinal flora. Therefore, the regulatory function on the intestinal flora is an important point of view and entry when evaluating dipeptide molecules with uric acid lowering activity. However, 400 kinds of common dipeptides are available, and it is not known which dipeptide molecules have xanthine oxidase inhibition and uric acid lowering activity.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a dipeptide which has the functions of inhibiting xanthine oxidase activity, reducing uric acid activity and regulating intestinal flora, and has the functions of relieving hyperuricemia and regulating intestinal flora, and an application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a dipeptide for relieving hyperuricemia and regulating intestinal flora, wherein the amino acid sequence of the dipeptide is as follows: cysteine-glutamic acid or/and lysine-glutamic acid.

The dipeptide is applied to the preparation of xanthine oxidase activity inhibitors.

The dipeptide is applied to the preparation of uric acid lowering medicines or medicines for adjuvant therapy of gout diseases.

The application of the dipeptide in the aspect of preparing intestinal flora regulator.

Compared with the prior art, the invention has the advantages that: the invention relates to a dipeptide capable of relieving hyperuricemia and regulating intestinal flora and application thereof, which is characterized in that firstly, 400 dipeptides obtained by randomly combining 20 common L-type amino acids in pairs are subjected to docking prediction with xanthine oxidase based on a Discovery studio platform and a molecular docking module; then 4 dipeptide molecules with potential inhibitory activity are selected to carry out in vitro enzyme activity inhibition test; selecting dipeptides with strongest in-vitro inhibitory activity, namely cysteine-glutamic acid (CE) and lysine-glutamic acid (KE), and evaluating the in-vivo uric acid reducing function of dipeptide molecules CE and KE by utilizing a hyperfructose-induced hyperuricemia mouse model; meanwhile, the CE and KE induced intestinal flora structure and function regulation is monitored by an intestinal flora metagenome sequencing technology, so that the function of regulating the intestinal flora is clear.

Drawings

FIG. 1 shows the results of in vitro measurement of xanthine oxidase inhibitory activities of four dipeptide molecules. A. Xanthine oxidase inhibitory activity of cysteine-glutamic acid (CE); B. xanthine oxidase inhibitory activity of lysine-glutamic acid (KE); C. xanthine oxidase inhibitory activity of lysine-methionine (KM); D. xanthine oxidase inhibitory activity of lysine-isoleucine (KI);

FIG. 2 is a graph showing the effect of dipeptide treatment on the glucose tolerance and insulin tolerance of high fructose-induced hyperuricemia mice. A. Serum glucose levels at different times in the glucose tolerance experiments with dipeptide CE and KE treatment; B. area under the curve in the glucose-time curve in the dipeptide CE and KE treatment versus glucose tolerance experiment; C. serum glucose levels at different times in the dipeptide KE treatment versus insulin resistance experiment; D. area under the curve in the glucose-time curve in the dipeptide KE treatment versus insulin resistance experiment;

FIG. 3 is a graph showing the effect of dipeptide CE and KE treatment on characteristic markers in serum and urine of mice with high fructose-induced hyperuricemia. A. Uric acid content in serum; B. urea nitrogen content in serum; C. uric acid content in urine; D. urea nitrogen content in urine;

FIG. 4 shows WB results for key proteins in uric acid synthesis, reabsorption and efflux pathways. A. WB results for ADA protein; B. WB results for GLUT9 protein; C. WB results for ABCG2 protein. The results are displayed as the ratio of the grey values of the target protein and the actin protein;

FIG. 5 shows the variation of the diversity of the intestinal flora alpha and beta of mice in the control group, model group, CE group and KE group. A. A Chao1 index; B. an observed speces index; C. shannon index; D. a simpson index; E. PCoA analysis results;

FIG. 6 is a graph showing the change in the phylum level (A) and class level (B) of the intestinal flora of mice in the control group, model group, CE group and KE group;

fig. 7 is a functional classification of intestinal flora in mice in the control group, model group, CE group and KE group at GO level (a) and KEGG level (B).

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

Detailed description of the preferred embodiment

High-throughput prediction and in vitro detection of xanthine oxidase inhibitory activity of dipeptide molecules

The method is based on Discovery Studio software, utilizes xanthine oxidase as a target, screens the assigning points of the interaction between 400 dipeptides and the xanthine oxidase, which are obtained by combining two common 20 amino acids, and comprises the following specific steps:

1. high throughput prediction of xanthine oxidase inhibitory activity of dipeptide molecules

(1) Establishment of a library of dipeptide ligands

Based on 20 common L-type amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), Chem3D software is used for drawing and generating three-dimensional structures of 400 dipeptides, MM2 force field optimization is carried out, and the three-dimensional structures are added into Discovery Studio software for high-throughput molecular docking screening;

(2) treatment of molecular docking acceptor xanthine oxidase

Selecting a xanthine oxidase crystal structure 1N5X to carry out molecular docking prediction, downloading the xanthine oxidase crystal structure 1N5X from a PDB database, preprocessing the 1N5X structure by using Discovery Studio software, and storing the structure in a pdbqt format for high-throughput molecular docking screening;

(3) molecular docking model prediction

Model prediction was performed using molecular docking (molecular docking) module of the Discovery Studio software. The docking center coordinates and the cassette size were set according to the primary ligand position parameters in 1N5X, with the other parameters being software defaults. 400 kinds of dipeptides were subjected to docking treatment with xanthine oxidase ((PDB database No. 1N 5X)) one by one, and an assignment was given according to the degree of spatial engagement between the ligand and the receptor, and the higher the assignment, the stronger the interaction between the ligand and the receptor, i.e., the stronger the xanthine oxidase inhibitory activity that may be present.

TABLE 1.400 assignment results of dipeptide to xanthine oxidase molecules

As a result, as shown in Table 1, the most highly assigned dipeptides were lysine-glutamic acid (KE, 90.04) and cysteine-glutamic acid (CE, 81.1).

2. In vitro xanthine oxidase inhibition activity assay of four dipeptides

The four dipeptides with the highest assignments were synthesized, cysteine-glutamic acid (CE, assignment 81.1), lysine-glutamic acid (KE, assignment 90.04), lysine-methionine (KM, assignment 70.96), and lysine-isoleucine (KI, assignment 77.74), respectively. The four dipeptides are respectively dissolved in sodium phosphate buffer solution (0.05 mol/L, pH 7.5) and diluted into dipeptide solutions (0.1-1.0 mg/mL) with different concentrations. Adding the dipeptide solutions with different concentrations into xanthine oxidase (7.5 × 10)^-8mol/L) was incubated at 37 ℃ for 30 min. Followed by addition of xanthine substrate (final concentration 5.0X 10)^–5mol/L), the absorbance of the mixture was measured every 30 s at room temperature using a spectrophotometer (295 nm). Using the formula: inhibitory Activity = (1-R/R0). times.100%, and IC was calculated from the inhibitory activity of the dipeptide against xanthine oxidase₅₀Value, wherein IC₅₀Lower values indicate stronger inhibitory activity of the dipeptide molecule against xanthine oxidase. Wherein R is the reaction rate of the xanthine oxidase catalyzed xanthine substrate after dipeptide addition, and R0 is the reaction rate of the xanthine oxidase catalyzed xanthine substrate without dipeptide addition.

As shown in FIG. 1, the dipeptides CE, KE, KM and KI inhibited the xanthine oxidase to different degrees, and the IC50 values were 1.65, 3.07, 3.16 and 20.31 mg/ml, respectively. Obtaining prediction assignments and experimental calculation IC by using Spearman correlation analysis and calculation₅₀The correlation coefficient between the values is-0.4, and certain negative correlation exists, so that the feasibility and the reliability of screening the xanthine oxidase inhibitor by using the Discovery Studio are proved.

Detailed description of the invention

Dipeptide CE and KE for relieving hyperuricemia

1. Intervention of dipeptide CE and KE on a mouse model of hyperuricemia induced by high fructose: the C57/B6J mice were randomized into 4 groups after 2 weeks of acclimation, namely a control group, a model group, a CE group and a KE group, with 5 mice per group. Wherein the control group is drinking normal drinking water, and the drinking water of other groups is added with 10% fructose. The control group and the model group are infused with equal dosage of normal saline, the CE group and the KE group are infused with 10 mg/kg/d dosage of dipeptide CE and KE respectively, 1 time per day and 12 weeks. Mouse feces and urine were collected at the end of 12 weeks, then the mouse eyes were bled and sacrificed, centrifuged at 3500 rp/min, serum was separated for 10 min and stored at-80 ℃.

2. Evaluation of the effects of the dipeptides CE and KE:

oral glucose tolerance test: the gastric glucose was gavaged at a dose of 1.5 g/kg, blood was drawn at intervals from the tail and the glucose content was determined using the kit.

Insulin resistance test: injecting insulin into abdominal cavity at a dose of 0.80U/kg, taking blood at intervals of tail, detecting glucose content by using the kit, and calculating corresponding parameters.

And (3) determining the hyperuricemia index: the kit is used for detecting the content of uric acid and urea nitrogen in serum and urine.

Western blot analysis: after extracting kidney/liver holoprotein, the samples were subjected to 10% SDS-PAGE electrophoresis and transferred to a difluoroethylene membrane to block non-specific binding sites. After the primary antibody was added and incubated overnight at 4 ℃, the secondary antibody was added and the results of the experiment were observed. Main detection targets of Western blot: the uric acid synthesis-related proteins Adenosine Deaminase (ADA) in the liver, glucose transporter 9 (GLUT 9) associated with uric acid reabsorption in the kidney, and uric acid efflux-related uric acid transporter (ABCG 2).

Given the possible effect of a long-term bolus fructose intake on the glucose and insulin resistance in mice, the mice were first subjected to an Oral Glucose Tolerance Test (OGTT). The results are shown in FIGS. 2A-2B, where blood glucose levels were highest in the model group and then gradually decreased in each group 20 min after oral administration of 2g/kg glucose. The area under the blood glucose curve (AUC) of the model group increased by 18% compared to the control group, whereas CE and KE interventions reduced it by 10.2% and 15.3%, respectively. Meanwhile, as the glucose tolerance of the mice treated with the dipeptide KE is closer to the level of the control group, the mice in the group are continuously subjected to intraperitoneal injection of 0.5U/kg dose of insulin for an Insulin Tolerance Test (ITT), and the results are shown in fig. 2C-2D, wherein the area under the blood glucose curve (AUC) is increased by 21% due to the decrease of the insulin tolerance of the mice by the intervention of the model group, and the area under the blood glucose curve (AUC) is decreased by 14.2% due to the intervention of the dipeptide KE.

The blood uric acid and blood urea nitrogen contents in the model group were increased by 47.2% and 77%, respectively, compared to the control group. Compared to the model group, CE treatment reduced blood uric acid and blood urea nitrogen levels by 6% and 18.6%, respectively, while KE treatment reduced blood uric acid and blood urea nitrogen levels by 18% and 18.1%, respectively. In addition, the uric acid and urea nitrogen contents in urine in the model group were respectively reduced by 44.6% and increased by 20.7% compared with the control group. Compared to the model group, CE treatment increased the blood uric acid and blood urea nitrogen content by 35.9% and 7%, respectively, while KE treatment increased the blood uric acid and blood urea nitrogen content by 42.2% and by 11.6%, respectively.

Subsequently, Western Blot technology is used for detecting protein expression levels of key proteins ADA, GLUT9 and ABCG2 in uric acid synthesis, reabsorption and excretion pathways. The results are shown in fig. 4A, where high fructose treatment increased ADA expression in the liver, while dipeptide CE and KE treatment reduced ADA expression to some extent, inhibiting uric acid synthesis, which is consistent with the prediction of xanthine oxidase inhibitory activity. In addition, high fructose treatment increased the expression of the key protein GLUT9 during reabsorption, dipeptide CE treatment had no effect on GLUT9 expression, and KE treatment further increased GLUT9 expression (fig. 4B). On the other hand, high fructose, although causing hyperuricemia, had an enhancing effect on the expression of exocrine protein ABCG2, whereas CE and KE treatment inhibited the excretion of uric acid instead (fig. 4C).

Detailed description of the preferred embodiment

Effect of dipeptides CE and KE on regulating intestinal flora

The feces of the mice of different treatment groups were subjected to metagenome high-throughput sequencing by NovaSeq 6000. The Chao1 index and the observed species index characterize the abundance (richness) of the intestinal flora (fig. 5A-5B), while the shannon index and the simpson index characterize the diversity (diversity) of the intestinal flora (fig. 5C-5D). Compared with a control group, the fructose modeling process increases the abundance and diversity of intestinal flora, the dipeptide CE and the KE treatment reduce the abundance of the intestinal flora, but the dipeptide CE and the KE treatment only reduce the shannon index in the diversity and have no significant influence on the Simpson index. Meanwhile, PCoA analysis found that the global structure of the intestinal flora was effectively modified by the high fructose modeling process to be completely different from the control group, while the dipeptide treatment restored the flora structure modified by the model group to a structure similar to the control group, but the impact of CE and KE on the intestinal flora was not completely consistent (fig. 5E).

In addition, the high fructose modeling process increased the abundance of the firmicites, Verrucomicrobia, Proteobacteria and Actinobacteria gates, but decreased the abundance of the bacteroidides gate; the effect of CE and KE treatment on the abundance at the phylum level was consistent, restoring varying Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria phyla abundances to the control level, while further increasing the abundance of Verrucomicrobia phyla (fig. 6A and table 2).

TABLE 2 Enterobacteriaceae phylum level abundance

Data are presented as mean ± standard deviation.

At the class level, the modeling process for high fructose increases the abundance of class Clostridia, Verrucomicrobiae, Deltaproteobacteria, Epsilonproteobacteria, Bacilli, Betaproteobacteria, Erysipelotrichia and Gamma, but decreases the abundance of class Bacteroides;

treatment of both CE and KE restored varying Bacteroides, Clostridia, Deltaproteobacteria, Epsilonproteobacteria, Bacilli and Gamma proteobacteria to control levels, further increasing the abundance of Verrucomicrobiae. In addition, KE processing decreased the abundance of Betaproteobacteria and Erysipelotrichia increased by the modeling process, but CE processing further exacerbated the changes in Betaproteobacteria and Erysipelotrichia (fig. 6B and table 3).

TABLE 3 Enterobacteriaceae class level abundance

Data are presented as mean ± standard deviation.

Meanwhile, the functional change of the intestinal flora is researched through a GO database and a KEGG database. As a result, it was found that in the GO classification, the molecular function (molecular function) and the biological process (biological process) function of the intestinal flora increased by the high fructose modeling treatment were decreased by the CE and KE treatments, and the cellular component (cellular component) function decreased by the modeling treatment was further decreased by the CE treatment and increased by the KE treatment (fig. 7A). In addition, in KEGG classification, the intestinal flora metabolism (metabolism) function decreased by the high fructose modeling process was increased by the CE and KE processes, the environmental information processing (cellular processes) and organic system (organic systems) functions increased by the modeling process were decreased by the CE and KE processes, and the genetic information processing (genetic information processing) function added by the modeling process was further increased by the CE and KE processes (fig. 7B).

The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Sequence listing

<110> Ningbo Green Jian Yao Kogyo Co., Ltd

<120> dipeptide capable of relieving hyperuricemia and regulating intestinal flora and application thereof

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Claims

1. The application of dipeptide in preparing xanthine oxidase activity inhibitor is characterized in that the amino acid sequence of the dipeptide is as follows: cysteine-glutamic acid.

2. The application of the dipeptide in the aspect of preparing uric acid lowering medicines or preparing gout disease adjuvant therapy medicines is characterized in that the amino acid sequence of the dipeptide is as follows: cysteine-glutamic acid.