A method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora

Abstract

The present invention belongs to the field of biomedicine technology, and in particular, relates to a method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora. The present invention takes the intestinal flora derived from the body as the research object, constructs an in vitro model with the group as the unit, maintains the diversity of the intestinal flora, studies the joint action of the microbial community, promotes more accurate intestinal flora-oriented research, and further co-cultivates the drug with the intestinal flora to obtain preliminary screening results through a fast and easy experimental method, which greatly shortens the time to obtain results, and then uses more precise detection methods to conduct in-depth research based on the obtained results, thereby improving the significance of screening. The present invention combines an in vitro model of intestinal flora with a xanthine oxidase activity inhibition experiment to provide a screening method for drugs that affect uric acid metabolism by targeting intestinal flora. The study of the relationship between intestinal flora and uric acid metabolism can also provide new clinical treatment targets for related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora. Background Art

[0002] There are a variety of symbiotic bacteria and other microorganisms in the intestines. In recent years, more and more studies have shown that the intestinal flora is closely related to the health and disease of the body. Drug therapy is becoming an important factor affecting the intestinal flora. There are complex interactions between microorganisms and drugs, that is, the intestinal flora can affect the pharmacological effects by metabolizing drugs, and drugs in turn will change the composition and function of intestinal microorganisms, potentially affecting the health of the host. As the intestinal microbial ecosystem and microbial pathways gradually become new targets for drug therapy, the theory of "pharmacomicrobiology" was proposed. "Pharmacomicrobiology" is to improve the efficacy of drugs by understanding the role of intestinal flora in drug therapy and promoting targeted intestinal flora therapy.

[0003] The intestinal flora is a complex microbial ecosystem that participates in many physiological metabolic activities of the body, including purine metabolism. When purine metabolism is disturbed, it will lead to increased uric acid. Uric acid is the end product of purine metabolism. Xanthine oxidase (XO) is a key rate-limiting enzyme in purine metabolism, which can oxidize hypoxanthine to xanthine or xanthine to uric acid. The imbalance of uric acid production and excretion will not only affect the normal physiological activities of the body, but also lead to increased uric acid levels in the blood, causing hyperuricemia. Long-term hyperuricemia can cause gout and be accompanied by a variety of diseases. Purine metabolism disorders in the intestine can also affect the intestinal barrier and promote intestinal inflammation. At present, the main clinical medicine for the treatment of excessive uric acid in the body is the following: (1) inhibiting uric acid production by inhibiting xanthine oxidase (allopurinol); (2) promoting uric acid excretion (probenecid); (3) promoting uric acid decomposition (purecil). Although these drugs can alleviate the disease, they also have significant side effects on the body. Therefore, it is very important in clinical treatment to develop effective methods with fewer side effects to lower uric acid levels.

[0004] At present, the research on drugs that affect uric acid metabolism still mainly relies on in vivo experiments on animal models. By promoting uric acid production, inhibiting uric acid excretion or inhibiting uricase activity, an animal model of hyperuricemia is established, and then drug treatment is used to screen uric acid-lowering drugs in vivo. In drug screening, not only is the experimental cycle long and the repeatability poor, but there are also problems such as high cost, complex operation, and low efficiency. Moreover, the current drug screening is mostly aimed at the inhibitory effect of the drug itself on enzyme activity. Different drugs have different in vivo metabolism, and there are relatively few studies on the interaction between drugs and flora after entering the intestine.

[0005] Intestinal flora can promote the catabolism and excretion of purine and uric acid, and plays a vital role in treatment. When kidney problems occur, the intestine becomes the main pathway for uric acid metabolism. Therefore, studying the relationship between intestinal flora and uric acid metabolism can provide new clinical treatment targets for related diseases. Based on this, we proposed a fast, simple and efficient in vitro screening method for uric acid-lowering drugs targeting intestinal flora based on an in vitro model of intestinal flora. Summary of the invention

[0006] The purpose of the present invention is to address the above problems, propose an in vitro screening method for uric acid-lowering drugs targeting intestinal flora based on an in vitro model of intestinal flora, and provide an in vitro screening method for drugs affecting xanthine oxidase activity targeting intestinal flora.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for targeting intestinal flora to screen drugs that affect xanthine oxidase activity comprises the following steps:

[0009] S1. Prepare in vitro model culture medium of intestinal flora;

[0010] S2, collect fresh fecal samples from mice for pretreatment and prepare fecal microbial suspension, all under anaerobic conditions;

[0011] S3, inoculating the fecal microorganisms prepared in step S2 into the culture medium described in step S1, adding the screening drug for co-incubation, and then centrifuging to collect the supernatant;

[0012] S4, using allopurinol as a positive control, determining the inhibitory activity of the drug for screening itself in step S3 and the supernatant obtained in step S3 on xanthine oxidase by ultraviolet spectrophotometry, eliminating the inhibitory effect of the drug for screening itself on xanthine oxidase, and screening out drugs that target intestinal flora and affect xanthine oxidase activity;

[0013] S5. Detect the difference in intestinal flora and the change in relative abundance of dominant flora in the sample after the drug screened out in step S4 is co-incubated with the fecal microorganisms prepared in step S2, and determine the effect of the drug for screening on the intestinal flora.

[0014] As a preferred technical solution of the present invention, the preparation method of the culture medium described in step S1 includes the following steps: adding peptone water, yeast extract, L-cysteine ​​hydrochloride, NaCl, K2HPO4, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, NaHCO3, hemin, vitamin K1, bile acid, chenodeoxycholic acid, and Tween 80 into water, wherein the water-insoluble substances are completely dissolved in the corresponding solvents and then added to the culture medium, ultrasonically dissolved, adjusting the pH value to 6.5±0.2, filtering and sterilizing, and storing in the dark.

[0015] As a preferred technical solution of the present invention, the concentrations of the substances in the culture medium are: peptone water 2.0 g / L, yeast extract 2.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L, NaCl 1.0 g / L, K2HPO4 0.4 g / L, KH2PO4 0.4 g / L, MgSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.1 g / L, NaHCO3 4.0 g / L, hemin 5 mg / L, vitamin K110 μL / L, bile acid 0.25 g / L, chenodeoxycholic acid 0.25 g / L, and Tween 80 2 mL / L.

[0016] As a preferred technical solution of the present invention, the method for preparing the fecal microbial suspension in step S2 is: adding the fecal sample to a phosphate buffer containing L-cysteine ​​hydrochloride and mixing, repeating the homogenization and centrifugation of the culture medium in step S1, combining the supernatant and centrifuging to collect the precipitate, and resuspending it using the culture medium. The ratio of the precipitate to the culture medium is 80:1 to 100:1 (mg / mL).

[0017] As a preferred technical solution of the present invention, the drug to be screened in step S3 is selected from one of Chinese herbal medicine extracts, chemically synthesized drugs, and biological preparations.

[0018] As a preferred technical solution of the present invention, the final inoculation concentration of the fecal microorganism in step S3 is 18% to 23% (w / v), the final concentration of the drug for screening is 95 to 100 μM, and the anaerobic co-incubation is carried out for 48 hours.

[0019] The present invention also provides application of the above method in screening uric acid-lowering drugs targeting intestinal flora.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention combines an in vitro model of intestinal flora with a xanthine oxidase activity inhibition experiment to provide a method for screening drugs that affect uric acid metabolism by targeting intestinal flora. This method makes up for the shortcomings of traditional drug research and provides a theoretical and practical basis for in vitro research on drug-microbial interactions, microbial metabolism, and drug screening.

[0022] (2) The present invention takes the microbiota derived from the body as the research object, constructs an in vitro model based on the group, maintains the diversity of the microbiota as much as possible, studies the joint effects of the microbial community, promotes more accurate microbiota-oriented research, and further co-cultivates the drug with the microbiota to obtain preliminary screening results through a rapid and easy experimental method, which greatly shortens the time to obtain results. Based on the obtained results, more precise detection methods are used for in-depth research, which improves the research significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a schematic diagram of the screening method.

[0024] Figure 2 This is a bar graph showing the relative inhibition rate of XO in the supernatant after co-incubation of rhein and intestinal flora in Example 1.

[0025] Figure 3 This is a bar graph showing the relative inhibition rate of rhein alone on XO in Example 1.

[0026] Figure 4 This is a graph showing changes in the abundance of intestinal microorganisms after treatment with rhein in Example 1.

[0027] Figure 5 This is a graph showing the difference in intestinal microbial species after treatment with rhein in Example 1.

[0028] Figure 6 The graph is a graph showing the changes in the relative abundance of dominant bacterial genera in the rhein treatment group in Example 1, t-test: *: P<0.05; **: P<0.01; ***: P<0.001.

[0029] Figure 7 This is a bar graph showing the relative inhibition rate of XO in the supernatant after co-incubation of 7,8-dihydroxyflavone with intestinal flora in Example 2 of the present invention.

[0030] Figure 8 This is a bar graph showing the relative inhibition rate of 7,8-dihydroxyflavone alone on XO in Example 2 of the present invention.

[0031] Fig. 9 This is a bar graph of the average relative abundance of intestinal flora after treatment with 7,8-dihydroxyflavone in Example 2 of the present invention, wherein: Fig. 9 a is the analysis of microbial abundance at the phylum level (Phylum); Fig. 9 b is the analysis of microbial abundance at the family level (Family); Fig. 9 c is the genus level microbial abundance analysis (Genus); Control: blank control group; 7,8-DHF: 7,8-dihydroxyflavone group.

[0032] Fig.10 This is a LEfSe analysis diagram of different species in the intestinal flora after treatment with 7,8-dihydroxyflavone in Example 2 of the present invention.

[0033] Fig.11 This is a graph showing the changes in relative abundance of bacterial genera in the 7,8-dihydroxyflavone-treated group in Example 2 of the present invention, t-test: *: P<0.05; **: P<0.01; ***: P<0.001. DETAILED DESCRIPTION

[0034] The technical solution 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0036] Example 1

[0037] In this implementation case, we choose rhein as the drug to be screened, and the specific screening method includes the following steps:

[0038] 1. Preparation of in vitro model culture medium for intestinal flora

[0039] Peptone water (2.0 g / L), yeast extract (2.0 g / L), L-cysteine ​​hydrochloride (0.5 g / L), NaCl (1.0 g / L), K2HPO4 (0.4 g / L), KH2PO4 (0.4 g / L), MgSO4·7H2O (0.1 g / L), CaCl2·2H2O (0.1 g / L), NaHCO3 (4.0 g / L), chlorinated Hemoglobin (5 mg / L), vitamin K1 (10 μL / L), bile acid (0.25 g / L), chenodeoxycholic acid (0.25 g / L), and Tween 80 (2 mL / L). The water-insoluble substances were completely dissolved in the corresponding solvents and then added to the culture medium. They were completely dissolved by ultrasound, and the pH value was adjusted to 6.5±0.2 (25°C) with dilute hydrochloric acid using a pH meter. The mixture was sterilized by filtration using a 0.22 μm filter and stored in the dark at 4°C for later use.

[0040] 2. Preparation of fecal microbial suspension

[0041] Collect fresh fecal samples from mice: The pretreatment of samples after collection was carried out in an anaerobic workstation (10% H2, 10% CO2 and 80% N2, 37°C). All materials and reagents required for the experiment were placed in the anaerobic workstation for pre-reduction treatment in advance. The specific steps are as follows:

[0042] (1) Collect 0.1 g of fresh mouse feces and place it in a sterile 2 mL centrifuge tube containing 250 μL of 0.1% L-cysteine ​​hydrochloride PBS solution.

[0043] (2) Rapidly transfer to an anaerobic workstation, open the tube cap, perform gas exchange, and vortex mix thoroughly until the mixture is nearly homogeneous.

[0044] (3) Add 750 μL of culture medium and vortex at maximum speed for 15 s; centrifuge at room temperature, 1000 rpm, for 30 s.

[0045] (4) Transfer the supernatant (about 750 μL) obtained by centrifugation into a clean, sterile 2 mL centrifuge tube A.

[0046] (5) Add 750 μL of culture medium to the remaining precipitate and vortex at maximum speed for 15 s; centrifuge at room temperature, 1000 rpm, for 30 s.

[0047] (6) Transfer the supernatant (about 750 μL) obtained by centrifugation to the 2 mL centrifuge tube A in step “4”.

[0048] (7) Add 750 μL of culture medium to the remaining precipitate and vortex at maximum speed for 15 s; centrifuge at room temperature, 1000 rpm, for 30 s.

[0049] (8) Transfer the supernatant (about 750 μL) obtained by centrifugation into a clean, sterile 2 mL centrifuge tube B.

[0050] (9) Add 750 μL of culture medium to the remaining precipitate and vortex at maximum speed for 15 s; centrifuge at room temperature, 1000 rpm, for 30 s.

[0051] (10) Transfer the supernatant obtained by centrifugation (about 750 μL) to the 2 mL centrifuge tube B in step "8" (at this point there should be 2 2 mL centrifuge tubes containing about 1.5 mL of supernatant).

[0052] (11) Centrifuge 2 mL centrifuge tube A containing approximately 1.5 mL of supernatant at room temperature at 4,000 × g for 5 min.

[0053] (12) Carefully remove and discard the supernatant after centrifugation. Transfer the liquid in 2 mL centrifuge tube B containing approximately 1.5 mL of supernatant to centrifuge tube A; centrifuge at 4,000 × g for 5 min at room temperature.

[0054] (13) Carefully remove and discard the supernatant after centrifugation and resuspend it in 1 mL of culture medium to obtain a mouse fecal microbial suspension.

[0055] 3. Drug treatment (co-incubation of rhein and intestinal flora)

[0056] (1) Immediately inoculate the treated mouse fecal microbial suspension into the optimized Mipro medium at a final inoculation concentration of 40% (w / v), and vortex to mix thoroughly.

[0057] (2) Drug treatment group: 500 μL of 40% (w / v) mouse fecal microbial suspension was pipetted into a 96-well plate; 100 μL of 1 mM rhein working solution and 400 μL of culture medium were added to each well.

[0058] (3) Positive control group: Pipette 500 μL of 40% (w / v) mouse fecal microbial suspension into a 96-well plate; add 100 μL of 1 mM allopurinol working solution and 400 μL of culture medium to each well.

[0059] (4) Blank control group: The mouse feces suspension treated with DMSO without adding any drug was used as the blank control. The final concentration of DMSO under the experimental conditions was 1%.

[0060] (5) Incubate the cells at 37°C under anaerobic conditions for 48 h.

[0061] 4. Determination of the inhibitory activity of xanthine oxidase in the supernatant after incubation of rhein with intestinal flora

[0062] (1) After incubation for 48 h, the samples of each treatment group were quickly removed and transferred to a clean, sterile 2 mL centrifuge tube. The tubes were centrifuged at 5,000 rpm for 10 min at room temperature. The supernatant of the samples was aspirated for testing, and the remaining samples were stored at -80 °C.

[0063] (2) Take 100 μL of the sample supernatant and add it to a 96-well plate. Then add 50 μL of xanthine oxidase (0.04 U / mL), mix by pipetting, and let stand at 37°C for 60 min. Add 50 μL of xanthine (400 μmol / L) to start the reaction. Mix by pipetting and be careful not to create bubbles.

[0064] (3) Use a multifunctional microplate reader to detect the change of absorbance at 290 nm over time within 20 minutes, and detect once every 120 seconds. The experimental results were statistically analyzed after three biological replicates. Plot the reaction time as the horizontal axis and the absorbance as the vertical axis and calculate the slope of the curve, that is, the reaction rate of the sample group is As; the reaction rate of the blank control group is Ac; the relative inhibition rate is calculated as follows:

[0065]

[0066] Results Analysis

[0067] The relative inhibition rate of the supernatant after co-incubation of rhein and bacteria on XO is calculated as follows: Figure 2 As shown, the relative inhibition rate of the supernatant after co-incubation of allopurinol and bacteria with the positive control group on xanthine oxidase was (60.15%±0.66)%. The relative inhibition rate of the supernatant after co-incubation of rhein and bacteria with the xanthine oxidase was (57.50%±4.51)%.

[0068] 5. The inhibitory effect of rhein alone on xanthine oxidase activity

[0069] (1) In the experiment, the positive control group was allopurinol, and the blank control group was culture medium containing 1% DMSO. 100 μL of rhein and allopurinol (100 μM) were added to a 96-well plate, and then 50 μL of xanthine oxidase (0.04 U / mL) was added. After pipetting and mixing, the plate was left to stand at 37°C for 60 min. 50 μL of xanthine (400 μmol / L) was added to start the reaction. Pipetting and mixing were performed to avoid bubbles.

[0070] (2) Use a multifunctional microplate reader to detect the change of absorbance at 290 nm over time within 20 minutes, and detect once every 120 seconds. The experimental results were statistically analyzed after three biological replicates. Plot the reaction time as the horizontal axis and the absorbance as the vertical axis and calculate the slope of the curve, that is, the reaction rate of the sample group is As; the reaction rate of the blank control group is Ac; the relative inhibition rate is calculated as follows:

[0071]

[0072] Results Analysis

[0073] The calculation results of the relative inhibition rate of rhein on XO are as follows: Figure 3 As shown, the relative inhibition rate of allopurinol alone in the positive control group on xanthine oxidase was (74.75%±4.63)%. Rhein alone had no significant inhibitory effect on xanthine oxidase, and its relative inhibition rate was 0%, which excluded the inhibitory effect of rhein itself on xanthine oxidase activity, and determined that the drug to be screened, rhein, was a drug that targeted the intestinal flora and affected the activity of xanthine oxidase.

[0074] 6. Detection of the regulatory effect of rhein on intestinal flora

[0075] 1 Experimental Materials

[0076] QIAamp PowerFecal Pro DNA Kit, Qiagen Biotechnology Co., Ltd.; SYBR qPCR MasterMix, Nanjing Novozymes Biotech Co., Ltd.

[0077] 2 Metagenomic Sequencing

[0078] (1) Library construction and sequencing

[0079] After the sample DNA was extracted using a DNA extraction kit and tested qualified, it was randomly sheared using an ultrasonic disruptor and then the library was constructed. After the construction was completed, Qubit 2.0 was used for preliminary quantification, the library was diluted, and then the insert fragments of the library were detected and the effective concentration of the library was quantified. After the library was tested qualified, the Illumina PE150 (2x150) platform was used for sequencing according to the effective concentration and the target data volume.

[0080] (2) Data preprocessing

[0081] The raw data obtained by sequencing (Raw Data) is preprocessed, including quality control and host sequence removal to obtain valid sequences (Clean Data) for subsequent analysis.

[0082] (3) Species and functional annotation and evaluation

[0083] Kraken2 software was used to annotate and classify valid sequences, and Bracken software was used to estimate the actual abundance of species in the samples. HUMAnN2 software was used to compare the quality control and host-free sequences with UniRef 90, and the annotation information and relative abundance table of each functional database were obtained based on the correspondence between UniRef 90 ID and each functional database.

[0084] (4) Data processing

[0085] The LEfSe (Linear discriminant analysis Effect Size, LEfSe) analysis method was used to perform significant difference analysis, and the data were classified and the influence of species with significant differences (i.e., LDA score) was evaluated based on linear discriminant analysis (LDA). The default LDA threshold was 2 to 4, and an LDA histogram was drawn.

[0086] Results Analysis

[0087] The results of changes in the abundance of intestinal flora microorganisms after drug treatment are as follows Figure 4 As shown:

[0088] Figure 4 a shows the changes in species abundance at the phylum level after rhein treatment. It can be seen from the figure that the flora mainly belongs to Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Compared with the blank control group, rhein treatment caused an increase in the abundance of Firmicutes and a decrease in the abundance of Bacteroidetes in the flora.

[0089] Figure 4 b shows the changes in species abundance of intestinal flora at the family classification level after rhein treatment. From the relative abundance of flora at the family level in each group, it can be seen that the detection rate of Enterobacteriaceae in each group is similar. Compared with the blank control group, the average relative abundance of Lactobacillaceae in the flora of the rhein-treated group increased, while the average relative abundance of Bacteroidaceae decreased.

[0090] Figure 4 c shows the changes in species abundance of intestinal flora at the genus classification level after rhein treatment. From the relative abundance of flora at the genus level in each group, it can be seen that the proportion of Escherichia in the Enterobacteriaceae family is relatively close in each group. The average relative abundance of the three genera of the Lactobacillaceae family (Lactobacillus, Limosilactobacillus and Ligilactobacillus) in the drug-treated group was higher than that in the blank control group.

[0091] Results of differential analysis of microbial species in the bacterial flora after drug treatment:

[0092] Depend on Figure 5It can be seen that compared with the blank control group, the abundance of Lactobacillus, Limosilactobacillus and Ligilactobacillus in the intestinal flora increased significantly after treatment with rhein, while the abundance of Phocaeicola and Parabacteroides in the flora decreased.

[0093] 3. Real-time fluorescence quantitative PCR was used to detect the changes in the relative abundance of regulatory bacteria in the bacterial community after drug treatment:

[0094] (1) The precipitate samples after drug and bacterial culture stored at -80°C were taken out and thawed naturally.

[0095] (2) Extract sample DNA according to the instructions of QIAamp○R PowerFecal○R Pro DNA Kit Fecal Genomic Extraction Kit.

[0096] (3) The quality of DNA extraction was tested by agarose gel electrophoresis, and the DNA concentration and purity were detected using a Nano Drop micro-volume UV spectrophotometer and stored in a -20°C refrigerator for future use.

[0097] (4) The SYBR fluorescent dye method was used for qPCR experiments. The total DNA of the samples that passed the quality inspection was diluted to 10 ng / μL with sterile ultrapure water as the DNA template for qPCR. Bacteroides, Bifidobacterium, Lactobacillus, Prevotella; Akkermansia, Enterococcus, and Enterobacteriaceae were detected. Three technical replicates were set for different primers for all detection templates, and each primer set included a non-template negative control.

[0098] (5) The 2-ΔΔCt method was used to analyze the relative difference fold, i.e., relative abundance, of the target gene of each bacterial genus. ΔΔCt = (Ct value of the target gene in the experimental group - Ct value of the reference gene in the experimental group) - (Ct value of the target gene in the control group - Ct value of the reference gene in the control group). The formula for calculating the difference fold of the relative expression of the target gene between the experimental group and the control group is:

[0099] Fold Change = 2 -ΔΔCt

[0100] Results Analysis

[0101] Real-time fluorescence quantitative PCR detection results Figure 6As shown in the results, after treatment with rhein, the relative abundance of Bacteroides (P<0.001), Prevotella (P<0.001), and Enterobacteriaceae (P<0.01) in the intestinal flora decreased significantly, and the relative abundance of Lactobacillus and Enterococcus increased significantly (P<0.001). There was no significant difference in the abundance of Bifidobacterium and Akkermansia muciniphila (P>0.05).

[0102] Example 2

[0103] In this implementation case, we selected 7,8-dihydroxyflavone as the drug to be screened, and the specific screening method and steps were the same as those in Example 1.

[0104] Results Analysis

[0105] The relative inhibition rate of XO in the supernatant after 7,8-dihydroxyflavone was co-incubated with intestinal flora is calculated as follows: Figure 7 As shown, the relative inhibition rate of the supernatant after co-incubation of allopurinol with intestinal flora in the positive control group on xanthine oxidase was (60.15%±0.66)%. The relative inhibition rate of the supernatant after co-incubation of 7,8-dihydroxyflavone with intestinal flora on xanthine oxidase was (50.57%±1.15)%.

[0106] The relative inhibition rate of 7,8-dihydroxyflavone on XO is calculated as follows: Figure 8 As shown, the relative inhibition rate of allopurinol alone in the positive control group on xanthine oxidase was (74.75%±4.63)%. 7,8-dihydroxyflavone alone had no significant inhibitory effect on xanthine oxidase, and its relative inhibition rate was (1.40%±1.80)%. It was determined that the drug to be screened, 7,8-dihydroxyflavone, was a drug that targeted intestinal flora and affected the activity of xanthine oxidase.

[0107] The results of changes in intestinal microbial abundance after 7,8-dihydroxyflavone treatment are as follows Fig. 9 As shown:

[0108] Fig. 9 a shows the changes in species abundance of intestinal flora at the phylum classification level after 7,8-dihydroxyflavone treatment. It can be seen from the figure that the flora mainly belongs to Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Compared with the blank control group, 7,8-dihydroxyflavone treatment caused an increase in the abundance of Firmicutes and a decrease in the abundance of Bacteroidetes in the flora.

[0109] Fig. 9b shows the changes in species abundance of intestinal flora at the family classification level after 7,8-dihydroxyflavone treatment. From the relative abundance of flora at the family level in each group, it can be seen that the detection rate of Enterobacteriaceae in each group is similar. Compared with the blank control group, the average relative abundance of Lactobacillaceae in the flora of the 7,8-dihydroxyflavone treatment group increased, and the average relative abundance of Bacteroidaceae decreased.

[0110] Fig. 9 c shows the changes in species abundance of intestinal flora at the genus classification level after treatment with 7,8-dihydroxyflavone. From the relative abundance of flora at the genus level in each group, it can be seen that the proportion of Escherichia in the Enterobacteriaceae family is relatively close in each group. The average relative abundance of the three genera of the Lactobacillaceae family (Lactobacillus, Limosilactobacillus and Ligilactobacillus) in the drug-treated group was higher than that in the blank control group.

[0111] Results of differential analysis of microbial species in the bacterial flora after treatment with 7,8-dihydroxyflavone:

[0112] Depend on Fig.10 It can be seen that compared with the blank control group, the abundance of Lactobacillus and Ligilactobacillus in the intestinal flora increased significantly after treatment with 7,8-dihydroxyflavone, while the abundance of Bacteroidales decreased significantly.

[0113] Real-time fluorescence quantitative PCR detection results Fig.11 As shown in the results, after 7,8-dihydroxyflavone treatment, the relative abundance of Bacteroides (P=0.004) in the intestinal flora decreased significantly, and the relative abundance of Lactobacillus (P=0.004) increased significantly, while there was no significant difference in the effects on Bifidobacterium, Prevotella, Akkermansia muciniphila, Enterococcus and Enterobacteriaceae (P>0.05).

[0114] In summary, rhein and 7,8-dihydroxyflavone alone have no significant inhibitory effect on xanthine oxidase, but inhibit xanthine oxidase activity by regulating intestinal flora. So far, the present invention has achieved the screening and effect verification of the effects of rhein and 7,8-dihydroxyflavone on xanthine oxidase activity by targeting intestinal flora using the constructed screening method.

[0115] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora, characterized in that: The steps include: S1. Prepare in vitro model culture medium of intestinal flora; S2, collect fresh fecal samples from mice for pretreatment and prepare fecal microbial suspension, all under anaerobic conditions; S3, inoculating the fecal microorganisms prepared in step S2 into the culture medium described in step S1, adding the drug to be screened for co-incubation, and then centrifuging to collect the supernatant; S4, using allopurinol as a positive control, determining the inhibitory activity of the drug for screening itself in step S3 and the supernatant obtained in step S3 on xanthine oxidase by ultraviolet spectrophotometry, eliminating the inhibitory effect of the drug for screening itself on xanthine oxidase, and screening out drugs that target intestinal flora and affect xanthine oxidase activity; S5, detecting the difference in intestinal flora and the change in relative abundance of dominant flora in the sample after the drug screened out in step S4 is co-incubated with the fecal microorganisms prepared in step S2, and determining the effect of the drug for screening on the intestinal flora; Step S1: The preparation method of the culture medium comprises the following steps: adding peptone water, yeast extract, L-cysteine ​​hydrochloride, NaCl, K2HPO4, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, NaHCO3, hemin chloride, vitamin K1, bile acid, chenodeoxycholic acid, and Tween 80 to water, wherein the water-insoluble substances are completely dissolved in the corresponding solvents and then added to the culture medium, ultrasonically dissolved, the pH value is adjusted to 6.5±0.2, filtered and sterilized, and stored in the dark; the concentrations of the substances in the culture medium are: peptone water 2.0 g / L, yeast extract 2.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L, NaCl 1.0 g / L, K2HPO4 0.4 g / L, KH2PO4 0.4 g / L, MgSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.1 g / L, NaHCO3 4.0g / L, hemin 5 mg / L, vitamin K1 10 μL / L, bile acid 0.25 g / L, chenodeoxycholic acid 0.25 g / L, Tween 802 mL / L; The drug is a uric acid lowering drug.

2. A method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora according to claim 1, characterized in that: The method for preparing the fecal microbial suspension in step S2 is: adding the fecal sample to a phosphate buffer containing L-cysteine ​​hydrochloride and mixing, repeating the homogenization and centrifugation of the culture medium in step S1, combining the supernatant and centrifuging to collect the precipitate, and resuspending it using the culture medium, and the ratio of the precipitate to the culture medium is 80:1 to 100:

1.

3. The method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora according to claim 1, characterized in that: The drug to be screened in step S3 is selected from a Chinese herbal medicine extract, a chemically synthesized drug, and a biological preparation.

4. The method for screening drugs that affect xanthine oxidase activity by targeting intestinal flora according to claim 1, characterized in that: The final inoculation concentration of the fecal microorganism inoculation in step S3 is 18% to 23% calculated by w / v, the final concentration of the drug for screening is 95 to 100 μM, and the anaerobic co-incubation is carried out for 48 hours.

5. Use of the method according to any one of claims 1 to 4 in screening uric acid-lowering drugs targeting intestinal flora.

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