CN113447579A - High-throughput screening method for tea xanthine oxidase antagonist - Google Patents
High-throughput screening method for tea xanthine oxidase antagonist Download PDFInfo
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- CN113447579A CN113447579A CN202110572934.2A CN202110572934A CN113447579A CN 113447579 A CN113447579 A CN 113447579A CN 202110572934 A CN202110572934 A CN 202110572934A CN 113447579 A CN113447579 A CN 113447579A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention relates to a high-throughput screening method of a xanthine oxidase antagonist in tea, which belongs to the technical field of screening of active ingredients in tea and specifically comprises the following steps: 1) preparing a tea extract; 2) incubating the tea extract with xanthine oxidase; 3) removing the compounds which are not combined with the xanthine oxidase by ultrafiltration; 4) dissociating to obtain a compound combined with xanthine oxidase; 5) identifying the structure of the compound; 6) compounds inhibit xanthine oxidase activity assays. The method has the advantages of simple and convenient operation, rapidness, low cost, high screening efficiency and the like.
Description
Technical Field
The invention belongs to the technical field of screening of active ingredients in tea, and particularly relates to a novel high-throughput screening method of a xanthine oxidase antagonist in tea.
Background
Hyperuricemia is an increase in blood uric acid caused by purine metabolic disorder and/or uric acid excretion disorder, and extracellular uric acid is in a supersaturated state. 5% -12% of patients with hyperuricemia develop gout, attack bones and joints, and easily accumulate the kidney and cardiovascular system. Hyperuricemia and gout have become a serious metabolic disease that endangers human health. Abnormalities in key enzymes in purine metabolism are the major cause of increased uric acid production. Xanthine oxidase is a key enzyme for promoting uric acid synthesis, and antagonists thereof may have the efficacy of reducing uric acid in human bodies. Therefore, the method has important significance in screening xanthine oxidase antagonists from natural plants.
Tea is one of the most popular drinks in the world and is also a natural health care product. According to statistics, the tea contains 450 compound components beneficial to human bodies. Researches show that the tea drinking can obviously reduce the uric acid level in the body, thereby relieving gout. However, since tea leaves are a complex mixture, the compounds with uric acid reducing effect in tea leaves are not clear at present, and the development of uric acid reducing health products and uric acid reducing drugs from tea leaves is hindered, so that the development of a method for screening the compounds with uric acid reducing effect from a complex mixture solution of tea leaves is urgently needed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a novel method for screening a xanthine oxidase antagonist in tea leaves at high flux. The method combines the affinity action of xanthine oxidase-tea compound, ultrafiltration and a metabonomics method based on liquid chromatogram-mass spectrum, and is used for screening the compound with the xanthine oxidase activity inhibition function in the tea at high flux.
The invention is realized by the following technical scheme:
the high-throughput screening method for the xanthine oxidase antagonist in the tea is characterized by comprising the following steps of:
1) preparing a tea extract;
2) incubating the tea extract with xanthine oxidase;
3) removing the compounds which are not combined with the xanthine oxidase by ultrafiltration;
4) dissociating to obtain a compound combined with xanthine oxidase;
5) screening compounds and identifying structures;
6) compounds inhibit xanthine oxidase activity assays.
Further, the preparation method of the tea extract in the step 1) comprises the following steps: extracting the compound in the tea leaves by using 0-99% methanol water solution or acetonitrile water solution at the extraction temperature of 10-90 ℃ for 2-120 minutes.
Further, the step of incubating the tea extract and xanthine oxidase in the step 2) comprises the following steps: mixing the tea extract and xanthine oxidase solution in equal volume, incubating in water bath at 15-45 deg.C for 0.1-48 hr, and setting as XOD group and control group: the tea extract and phosphate buffer solution with pH 6.8 were mixed in equal volumes, and then incubated in water bath at the same incubation temperature and incubation time as the XOD group.
Further, the method for removing the compounds which are not combined with the xanthine oxidase by ultrafiltration in the step 3) comprises the following steps: after the incubation is finished, carrying out centrifugal ultrafiltration on the mixed solution of the tea extract and the xanthine oxidase solution, wherein the specification of an ultrafiltration tube is 3 KD-500 KD, and the centrifugal rotation speed is 13000-15000 g; and adding deionized water to further clean the compounds which are not combined with xanthine oxidase for 2-10 times, and discarding the cleaning solution.
Further, the dissociation method for obtaining the compound bound to xanthine oxidase in the step 4) comprises the following steps: adding 0.5-10mL of methanol aqueous solution into the ultrafiltration tube cleaned by the deionized water to dissociate the compound combined with the xanthine oxidase into the methanol aqueous solution, and further performing centrifugal ultrafiltration at the centrifugal rotation speed of 13000-15000 g; the aqueous methanol solution was collected.
Further, the compound screening and structure identification in the step 5) are specifically as follows: carrying out high-resolution liquid chromatography-mass spectrometry analysis on the methanol aqueous solution collected in the step 4), screening out a compound with a ratio of >1.5 in the XOD group solution to the control group solution and with a Student's t-test p of <0.05 according to the mass spectrometry information of the accurate molecular weight of the compound, and carrying out structure identification.
Further, the method for determining the xanthine oxidase activity inhibition activity of the compound in the step 6) comprises the following steps: and (3) preparing a compound monomer from the compound screened in the step 5) by purchasing a standard sample or separating and purifying the compound monomer from tea leaves, preparing a solution with the concentration of 20 mu mol/L, and measuring the inhibition rate of the compound on the xanthine oxidase activity by adopting a xanthine oxidase activity detection kit.
Further, the proportion of the methanol in the methanol water solution is 10-100%.
Further, the high-resolution liquid chromatography-mass spectrometry analysis instrument is UPLC-Q-active; the liquid chromatography conditions were: acquity UPLC BEH C18 column, 100X 2.1mm, 1.7 μm, mobile phase 0.1% formic acid/water solution and acetonitrile, mobile phase gradient set up as follows: 0 min, 10% B; 3 minutes, 15% B; 15 min, 65% B; 19 minutes, 95% B; 22 minutes, 95% B; 22.5 min, 10% B; 26 minutes, 10% B; the flow rate is 0.35 ml/min, and the sample injection amount is 5 microliter; the mass spectrometry conditions used were: an electrospray ion source is adopted, the ionization mode is positive ions, the voltage is 3kV, the heating temperature of the ion source is set to be 600 ℃, and the taper hole airflow and the desolventizing airflow are respectively set to be 50 liters/hour and 600 liters/hour.
Compared with the prior art, the invention has the following beneficial effects:
1) the tea extract can be directly used for incubation with xanthine oxidase to screen active compounds with xanthine oxidase inhibition capability, a previous separation and purification step of compound monomers in tea is not needed, and the workload is greatly saved;
2) in the process of screening the active compounds with xanthine oxidase inhibition capability, all compounds in the tea extract compete with the xanthine oxidase together for binding, and the active compounds with strong binding capability to the xanthine oxidase are directly screened out;
3) the method has the advantages of simple and rapid operation and low cost, and has wide application prospect in the field of screening and researching of active ingredients of tea.
Drawings
FIG. 1 is a schematic diagram of the screening method of the present invention.
Detailed Description
The following detailed description and specific operation procedures are given in conjunction with the accompanying drawings to provide a better understanding of the present invention, and the present invention is not limited to the following embodiments.
Example 1
1) Preparing a green tea extracting solution: extracting the compound in the tea leaves by using a methanol aqueous solution with the concentration of 2%, wherein the extraction temperature is 70 ℃, and the extraction time is 30 minutes;
2) co-incubation of green tea extract with xanthine oxidase: mixing the green tea extract and xanthine oxidase solution in equal volume, and incubating in water bath at 37 deg.C for 0.5 hr to obtain XOD group (parallel experiment repetition number n is 6); mixing the green tea extract with an equal volume of phosphate buffer (pH 6.8), and incubating in water bath at 37 deg.C for 0.5 hr as a control (parallel experiment repeated for n 6);
3) ultrafiltration removed compounds not bound to xanthine oxidase: after the incubation is finished, transferring the mixed solution of the green tea extract and the xanthine oxidase solution into an ultrafiltration centrifugal tube for centrifugal ultrafiltration, wherein the specification of the ultrafiltration tube is 5KD, and the centrifugal rotation speed is 15000 g; then adding deionized water to further clean the compounds which are not combined with xanthine oxidase for 4 times, and discarding the cleaning solution;
4) dissociation yields compounds that bind to xanthine oxidase: adding 2mL of 50% methanol aqueous solution into the ultrafiltration tube cleaned with deionized water, dissociating the compound combined with xanthine oxidase into 50% methanol aqueous solution, and further performing centrifugal ultrafiltration at 15000g centrifugal speed; collecting the methanol aqueous solution after ultrafiltration;
5) and (3) identifying the structure of the compound: and (3) carrying out high-resolution liquid chromatography-mass spectrometry analysis on the collected XOD group and the control group methanol aqueous solution, and carrying out structural identification on the compound which is remarkably increased (the ratio is greater than 1.5, Student's t-test p is less than 0.05) in the XOD group solution compared with the control group according to the information of the compound such as accurate molecular weight (primary mass spectrum), accurate secondary mass spectrum and the like. The high-resolution liquid chromatography-mass spectrometry instrument is UPLC-Q-active; the liquid chromatography conditions were: acquity UPLC BEH C18 column, 100X 2.1mm, 1.7 μm, mobile phase 0.1% formic acid/water solution and acetonitrile, mobile phase gradient set up as follows: 0 min, 10% B; 3 minutes, 15% B; 15 min, 65% B; 19 minutes, 95% B; 22 minutes, 95% B; 22.5 min, 10% B; 26 minutes, 10% B; the flow rate is 0.35 ml/min, and the sample injection amount is 5 microliter; the mass spectrum conditions are as follows: an electrospray ion source is adopted, the ionization mode is positive ions, the voltage is 3kV, the heating temperature of the ion source is set to be 600 ℃, and the taper hole airflow and the desolventizing airflow are respectively set to be 50 liters/hour and 600 liters/hour. The main compounds identified in the obtained green tea that bind to xanthine oxidase are shown in table 1;
6) compound inhibition of xanthine oxidase activity assay: the compounds selected in the above steps are prepared into compound monomers by purchasing standard samples or separating and purifying from tea leaves, and prepared into a solution with the concentration of 20 mu mol/L, and the inhibition rate of the compounds on the xanthine oxidase activity is determined by adopting a xanthine oxidase activity detection kit (purchased from Sigma company, with the product number of MAK078-1 KT). The measurement results show that the inhibition rate of the xanthine oxidase activity by the compound monomer selected in the above procedure in green tea is shown in table 1.
TABLE 1 inhibition of xanthine oxidase by selected compounds from green tea and inhibition thereof
Example 2
1) Preparing a black tea extracting solution: extracting the compound in the tea leaves by using 1.5 percent methanol water solution at the extraction temperature of 70 ℃ for 40 minutes;
2) co-incubating the black tea extract with xanthine oxidase: mixing black tea extract and xanthine oxidase solution in equal volume, and incubating in water bath at 37 deg.C for 0.5 hr to obtain XOD group (parallel experiment repetition number n is 6); mixing black tea extract with equal volume of phosphoric acid buffer solution (pH 6.8), and incubating in water bath at 37 deg.C for 0.5 hr as control group (parallel experiment repetition number n 6);
3) ultrafiltration removed compounds not bound to xanthine oxidase: after incubation, transferring the mixed solution of the black tea extract and the xanthine oxidase solution into an ultrafiltration centrifugal tube for centrifugal ultrafiltration, wherein the specification of the ultrafiltration tube is 10KD, and the centrifugal rotation speed is 15000 g; then adding deionized water to further clean the compounds which are not combined with xanthine oxidase for 4 times, and discarding the cleaning solution;
4) dissociation yields compounds that bind to xanthine oxidase: adding 2mL of 50% methanol aqueous solution into the ultrafiltration tube cleaned with deionized water, dissociating the compound combined with xanthine oxidase into 50% methanol aqueous solution, and further performing centrifugal ultrafiltration at 15000g centrifugal speed; collecting the methanol aqueous solution after ultrafiltration;
5) and (3) identifying the structure of the compound: and (3) carrying out high-resolution liquid chromatography-mass spectrometry analysis on the collected XOD group and the control group methanol aqueous solution, and carrying out structural identification on the compound which is remarkably increased (the ratio is greater than 1.5, Student's t-test p is less than 0.05) in the XOD group solution compared with the control group according to the information of the compound such as accurate molecular weight (primary mass spectrum), accurate secondary mass spectrum and the like. The high-resolution liquid chromatography-mass spectrometry instrument is UPLC-Q-active; the liquid chromatography conditions were: acquity UPLC BEH C18 column, 100X 2.1mm, 1.7 μm, mobile phase 0.1% formic acid/water solution and acetonitrile, mobile phase gradient set up as follows: 0 min, 10% B; 3 minutes, 15% B; 15 min, 65% B; 19 minutes, 95% B; 22 minutes, 95% B; 22.5 min, 10% B; 26 minutes, 10% B; the flow rate is 0.35 ml/min, and the sample injection amount is 5 microliter; the mass spectrum conditions are as follows: an electrospray ion source is adopted, the ionization mode is positive ions, the voltage is 3kV, the heating temperature of the ion source is set to be 600 ℃, and the taper hole airflow and the desolventizing airflow are respectively set to be 50 liters/hour and 600 liters/hour. The main compounds identified in black tea that bind to xanthine oxidase are shown in table 2.
(6) Compound inhibition of xanthine oxidase activity assay: the compounds selected in the above steps are prepared into compound monomers by purchasing standard samples or separating and purifying from tea leaves, and prepared into a solution with the concentration of 20 mu mol/L, and the inhibition rate of the compounds on the xanthine oxidase activity is determined by adopting a xanthine oxidase activity detection kit (purchased from Sigma company, with the product number of MAK078-1 KT). The measurement results show that the inhibition ratio of the xanthine oxidase activity by the compound monomer selected in the above steps in black tea is shown in table 2.
TABLE 2 main compounds selected from Black tea having inhibitory effect on xanthine oxidase and their inhibitory rates
Claims (9)
1. A high-throughput screening method of a xanthine oxidase antagonist in tea leaves is characterized in that
The method comprises the following steps:
preparing a tea extract;
2) incubating the tea extract with xanthine oxidase;
3) removing the compounds which are not combined with the xanthine oxidase by ultrafiltration;
4) dissociating to obtain a compound combined with xanthine oxidase;
5) screening compounds and identifying structures;
6) compounds inhibit xanthine oxidase activity assays.
2. The method for high throughput screening of tea xanthine oxidase antagonists according to claim 1, wherein the preparation method of the tea extract in step 1) comprises: extracting the compound in the tea leaves by using 0-99% methanol water solution or acetonitrile water solution at the extraction temperature of 10-90 ℃ for 2-120 minutes.
3. The method for screening the high throughput of the tea xanthine oxidase antagonist according to claim 1, wherein the incubating step of the tea extract and the xanthine oxidase in step 2) comprises the following steps: mixing the tea extract and xanthine oxidase solution in equal volume, incubating in water bath at 15-45 deg.C for 0.1-48 hr, and setting as XOD group and control group: the tea extract and phosphate buffer solution pH =6.8 were mixed in equal volumes and then incubated in a water bath at the same incubation temperature and incubation time as the XOD group.
4. The method for screening tea leaf xanthine oxidase antagonist in high throughput according to claim 1, wherein the ultrafiltration in step 3) to remove compounds that do not bind to xanthine oxidase comprises: after the incubation is finished, carrying out centrifugal ultrafiltration on the mixed solution of the tea extract and the xanthine oxidase solution, wherein the specification of an ultrafiltration tube is 3 KD-500 KD, and the centrifugal rotation speed is 13000-g(ii) a And adding deionized water to further clean the compounds which are not combined with xanthine oxidase for 2-10 times, and discarding the cleaning solution.
5. The method for screening tea leaf xanthine oxidase antagonist in high throughput according to claim 1, wherein the dissociation method for obtaining the compound binding to xanthine oxidase in step 4) comprises: adding 0.5-10mL methanol water solution into the ultrafiltration tube cleaned with deionized water to dissociate the compound combined with xanthine oxidase into the methanol water solution, and further performing centrifugal ultrafiltration at 13000-15000 centrifugal speedg(ii) a The aqueous methanol solution was collected.
6. The method for high throughput screening of tea xanthine oxidase antagonists according to claim 1, wherein the compound screening and structure identification in step 5) comprises: carrying out high-resolution LC-MS analysis on the methanol aqueous solution collected in the step 4), and screening out the ratio of the XOD group solution to the control group solution according to the mass spectrum information of the accurate molecular weight of the compound>1.5，Student’s tChecking p<0.05 and carrying out structural identification.
7. The method for screening tea leaf xanthine oxidase antagonist in high throughput according to claim 1, wherein the assay method for the compound inhibiting xanthine oxidase activity in step 6) is: and (3) preparing a compound monomer from the compound screened in the step 5) by purchasing a standard sample or separating and purifying the compound monomer from tea leaves, preparing a solution with the concentration of 20 mu mol/L, and measuring the inhibition rate of the compound on the xanthine oxidase activity by adopting a xanthine oxidase activity detection kit.
8. The method for high throughput screening of tea xanthine oxidase antagonists according to claim 5, wherein the methanol in the aqueous methanol solution is 10% to 100%.
9. The method for high throughput screening of tea xanthine oxidase antagonists according to claim 6, wherein the high resolution liquid chromatography-mass spectrometry is performed using UPLC-Q-active; the liquid chromatography conditions were: acquity UPLC BEH C18 column, 100X 2.1mm, 1.7 μm, mobile phase 0.1% formic acid/water solution and acetonitrile, mobile phase gradient set up as follows: 0 min, 10% B; 3 minutes, 15% B; 15 min, 65% B; 19 minutes, 95% B; 22 minutes, 95% B; 22.5 min, 10% B; 26 minutes, 10% B; the flow rate is 0.35 ml/min, and the sample injection amount is 5 microliter; the mass spectrometry conditions used were: an electrospray ion source is adopted, the ionization mode is positive ions, the voltage is 3kV, the heating temperature of the ion source is set to be 600 ℃, and the taper hole airflow and the desolventizing airflow are respectively set to be 50 liters/hour and 600 liters/hour.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102095825A (en) * | 2010-12-08 | 2011-06-15 | 中国科学院长春应用化学研究所 | Method for screening xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry |
| CN103558306A (en) * | 2013-10-31 | 2014-02-05 | 中南大学 | Composite for screening xanthine oxidase inhibitor and application method thereof |
| CN110840930A (en) * | 2019-12-11 | 2020-02-28 | 中北大学 | Method for extracting xanthine oxidase inhibitor from perilla leaves with assistance of ultrasonic waves |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102095825A (en) * | 2010-12-08 | 2011-06-15 | 中国科学院长春应用化学研究所 | Method for screening xanthine oxidase inhibitor by ultra performance liquid chromatography and mass spectrometry |
| CN103558306A (en) * | 2013-10-31 | 2014-02-05 | 中南大学 | Composite for screening xanthine oxidase inhibitor and application method thereof |
| CN110840930A (en) * | 2019-12-11 | 2020-02-28 | 中北大学 | Method for extracting xanthine oxidase inhibitor from perilla leaves with assistance of ultrasonic waves |
Non-Patent Citations (2)
| Title |
|---|
| LIANGLIANG LIU等: "Analysis of Xanthine Oxidase Inhibitors from Puerariae flos Using Centrifugal Ultrafiltration Coupled with HPLC-MS", 《J. BRAZ. CHEM. SOC.》 * |
| 汪生 等: "基于超高效液相色谱-高分辨质谱联用的非靶向代谢组学分析普洱生茶和普洱熟茶醇溶性物质组成差异", 《安徽农业科学》 * |
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