阅读说明：本技术 一种基于改性纤维富集的抗痛风药物筛选方法 (Anti-gout drug screening method based on modified fiber enrichment ) 是由 陶益 陈林 江恩赐 朱菲 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种基于改性纤维富集的抗痛风药物筛选方法,在聚偏二氟乙烯纤维表面依次覆盖功能化聚多巴胺、聚乙烯亚胺和黄嘌呤氧化酶得到靶标键合纤维,然后将靶标键合纤维和待筛选的药用植物提取物混合、孵育；孵育结束后先用PBS缓冲液清洗,再用变性清洗液进行变性洗脱,然后对变性洗脱物进行液质分析,获取靶标结合物的相关质谱信息,同时采用表面等离子共振技术分析靶标-小分子的相互作用,获取KD值,筛选出抗痛风成分。本发明提供的筛选方法能够用于药用植物中抗痛风成分的筛选,与传统植物化学分离筛选方法相比,可显著提高筛选效率、缩短筛选时间,可重复使用,具有快速、高效、准确、重现性好等优点。(The invention discloses a method for screening anti-gout drugs based on modified fiber enrichment, which comprises the steps of sequentially covering the surface of polyvinylidene fluoride fiber with functionalized polydopamine, polyethyleneimine and xanthine oxidase to obtain target bonding fiber, and then mixing and incubating the target bonding fiber and a medicinal plant extract to be screened; and after incubation, washing with PBS buffer solution, performing denaturing elution with a denaturing washing solution, performing liquid mass analysis on the denatured eluate to obtain related mass spectrum information of the target conjugate, analyzing the interaction of the target and the small molecule by adopting a surface plasmon resonance technology to obtain a KD value, and screening out the anti-gout component. The screening method provided by the invention can be used for screening anti-gout components in medicinal plants, can obviously improve the screening efficiency and shorten the screening time compared with the traditional plant chemical separation screening method, can be repeatedly used, and has the advantages of rapidness, high efficiency, accuracy, good reproducibility and the like.)

1. A method for screening anti-gout drugs based on modified fiber enrichment is characterized by comprising the following steps: the method comprises the following steps: performing biomimetic modification on the surface of polyvinylidene fluoride fiber by utilizing polydopamine and polyethyleneimine, bonding a target on the surface of the polyethyleneimine, mixing and incubating the target bonding fiber and the medicinal plant extract; and after incubation, washing with PBS buffer solution, performing denaturing elution with a denaturing washing solution, performing liquid mass analysis on the denatured eluate to obtain related information of the target conjugate, and separating and obtaining the specific binding ligand from the medicinal plants.

2. The method of claim 1, wherein: the denatured cleaning solution is acetonitrile water solution or methanol water solution.

3. The method of claim 1, wherein: after the incubation is finished, washing for 3 times by using PBS buffer solution, and denaturing the washing solution into methanol aqueous solution with the volume concentration of 70%.

4. The method of claim 1, wherein: incubating the target binding fiber and the extract of the medicinal plant to be screened at 35 deg.C for 20 min.

5. The method of claim 1, wherein: the target bonded fiber and the extract of the medicinal plant are mixed, and a PBS buffer solution is added to make the ionic strength of the solution be 333-337mM and the pH value be 6.7-6.9 for incubation.

6. The method of claim 1, wherein: the method comprises the following steps:

(1) immersing a certain amount of fibers into Tris-HCl buffer solution, and carrying out ultrasonic treatment for half an hour to activate the surfaces of the fibers;

(2) adding polydopamine solution into the activated fiber obtained in the step (1), shaking and mixing uniformly, washing for three times after stirring overnight, then sequentially suspending and stirring in phosphate buffer solution of polyethyleneimine and xanthine oxidase for a period of time, and washing for several times to obtain xanthine oxidase bonded fiber;

(3) extracting herba plantaginis with boiling water under reflux to obtain extractive solution, filtering, centrifuging, and collecting supernatant to obtain herba plantaginis crude extract;

(4) and (3) inserting the crude extract of the plantain herb into the xanthine oxidase bonded fiber prepared in the step (2), adding PBS buffer solution, uniformly mixing on an oscillator, wherein the ionic strength of the solution is 333-337mM and the pH value is 6.7-6.9, incubating for 20min at 35 ℃, then pulling out the xanthine oxidase bonded fiber, washing for 3 times by using 12mL incubation buffer solution, performing denaturation elution by using 2mL of 70% methanol-water solution, and performing liquid quality analysis and surface plasmon resonance analysis on the eluate.

7. The method of claim 6, wherein: the parameters of the liquid mass analysis are ESI negative ion mode.

Technical Field

The invention relates to a method for screening anti-gout active ingredients from medicinal plant extracts, in particular to an affinity solid-phase microextraction method which has high screening efficiency and environmental protection and can synchronously carry out enrichment and identification on the active ingredients, and has important significance for quickly screening the anti-gout active ingredients.

Background

The traditional screening mode of the active ingredients of the medicinal plants mainly comprises two steps: firstly, monomer compounds are separated through means of extraction, column chromatography, purification and the like, and secondly, activity test is carried out on the monomer compounds. The whole screening process has the defects of complexity, time consumption, consumption of a large amount of organic solvents, environmental friendliness and the like. The solid phase micro-extraction technology is a new sample pretreatment enrichment technology, and is often combined with gas chromatography-mass spectrometry to detect volatile components and analyze harmful chemical substances such as polycyclic aromatic hydrocarbons, microcystins and environmental pollutants in chemical environments such as water, soil, atmosphere and the like. The affinity solid phase microextraction technology is characterized by that on the surface of modified fibre a target is fixed, so that the target extraction fibre can be prepared, after the target extraction fibre is combined with ligand, the fibre can be taken out, and the non-specific combined component can be separated, then the combined ligand can be eluted by means of eluent. Currently, a number of fiber surface modification techniques have been developed to immobilize targets. For example, polydopamine exhibits strong adhesion properties to many materials through spontaneous oxidative self-polymerization, can be used for immobilization reactions by michael addition or by forming a schiff base to link various biomolecules such as proteins and enzymes, and thus, polydopamine is widely used for biomimetic surface modification.

Polyethyleneimine is a cationic polymer comprising amine groups and two carbon aliphatic CH groups₂CH₂Spacer, a negatively charged target, readily binds to fibers coated with positively charged polyethyleneimine. Compared with the traditional method, the affinity solid phase micro-extraction technology has the advantages of high speed, high efficiency, low organic reagent consumption, environmental protection and the like. The xanthine oxidase is a main target of anti-gout drugs, and no report exists on a method for screening xanthine oxidase inhibitors by applying an affinity solid phase microextraction-ultra-high performance liquid phase-mass spectrometry combined technology. The active ingredient enrichment technology based on the modified fiber has the advantages of short operation time, less consumption of organic reagents, high enrichment efficiency and the like.

Disclosure of Invention

The invention aims to provide a method for screening anti-gout drugs based on modified fiber enrichment.

In order to realize the purpose, the invention adopts the technical scheme that:

a method for screening anti-gout drugs based on modified fiber enrichment comprises the following steps: sequentially covering the surface of polyvinylidene fluoride fiber with functional polydopamine, polyethyleneimine and xanthine oxidase to obtain target bonding fiber, and then mixing and incubating the target bonding fiber and the medicinal plant extract to be screened; and after incubation, washing with PBS buffer solution, performing denaturing elution with a denaturing washing solution, performing liquid mass analysis on the denatured eluate to obtain related mass spectrum information of the target conjugate, analyzing the interaction of the target and the small molecule by adopting a surface plasmon resonance technology to obtain a KD value, and screening out the anti-gout component.

Preferably, the denatured washing liquid is an acetonitrile aqueous solution or a methanol aqueous solution.

Preferably, after the incubation is finished, the cells are washed for 3 times by using PBS buffer, and the denatured and washed solution is 70% methanol aqueous solution by volume concentration.

Preferably, the target binding fiber and the extract of the medicinal plant to be screened are incubated at 35 ℃ for 20 min.

Preferably, the target binding fiber and the extract of the medicinal plant to be screened are mixed, and a PBS buffer is added to make the pH of the incubation solution be 6.7-6.9.

Preferably, the target binding fiber and the drug to be screened are mixed and added with PBS buffer solution to make the ionic strength of the incubation solution 333-337 mM.

The method for screening the anti-gout drugs based on the modified fiber enrichment comprises the following steps:

(1) taking a bundle of polyvinylidene fluoride fibers, and carrying out ultrasonic treatment for half an hour by using an equal volume of 10mM Tris-hydrochloride buffer solution (pH 8.5) to obtain activated fibers for later use;

(2) and (2) adding 80mg of dopamine into the activated fiber obtained in the step (1), and stirring the solution overnight to obtain the polydopamine-coated fiber. The polydopamine-coated fibers were washed 3 times with ultrapure water and then immersed in 12mL of polyethyleneimine (20 mg/mL). Stirring the solution for 2 hours to obtain polyethyleneimine covered fibers; immersing the polyethyleneimine-covered fibers in 12mL of a xanthine oxidase solution (1mg/mL), and stirring the solution for 3 hours to obtain xanthine oxidase-covered fibers;

(3) weighing herba plantaginis, adding distilled water, reflux-extracting to obtain herba plantaginis extractive solution, filtering, collecting filtrate, centrifuging, and collecting supernatant to obtain herba plantaginis crude extract;

(4) adding the plantain herb extracting solution into the xanthine oxidase covered fiber prepared in the step (3), adding PBS buffer solution, uniformly mixing on an oscillator to ensure that the ionic strength of the solution is 333-337mM and the pH value is 6.7-6.9, incubating for 20min at 35 ℃, then taking out the incubated fiber, firstly washing for 3 times by using the PBS buffer solution, then performing denaturing elution by using 70% methanol aqueous solution, performing liquid-mass analysis on the denatured eluent, and performing surface plasmon resonance verification on the active small molecules.

In the method for screening the anti-gout drugs of the medicinal plants based on fiber enrichment, the liquid quality parameter of the liquid quality analysis is in an ESI negative ion mode.

Some abbreviations that have been used herein have the following meanings: PBS phosphate buffer salt; KD value: a dissociation constant; ESI: electrospray ionization; tris is Tris hydroxymethyl aminomethane.

Has the advantages that: the method for screening the anti-gout drugs of the medicinal plants based on the enrichment of the modified fibers has the following advantages:

the method for screening the anti-gout drugs of the medicinal plants based on the enrichment of the modified fibers has reasonable process design, and can screen out the optimal incubation temperature, incubation time, pH of incubation solution, ionic strength and proportion of denatured cleaning solution through a large number of experiments.

The invention is mainly applied to screening the anti-gout active ingredients in the medicinal plants, has reasonable design, high efficiency and perfect structure of the provided screening and evaluating system, can be used for identifying the anti-gout ingredients in the medicinal plants, has simple and economical equipment, and is suitable for exploring the identification performance of more targets in various medicinal plants. Compared with the traditional plant chemical method, the method obviously improves the screening efficiency, shortens the screening time, can be recycled, and is green and environment-friendly.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.

Reagents used in the following examples:

xanthine oxidase (EC: 1.1.3.22), anti-xanthine oxidase antibody (Bioss Biotechnology, Inc.), goat anti-rabbit antibody (Ronga Biotechnology, Inc.), allopurinol (Alatin Biotechnology, Inc.), seven standards: phillyrin C, isoplantaginide, plantaginide, luteolin-7-O-diglucuronide, isoacteoside, verbascoside, scutellarin, baicalin A, hispidulin-7-O-beta-D-glucuronide (Vickqi Biotech Co., Ltd.), and vanillic acid (Yuanye Biotech Co., Ltd.). Polyvinylidene fluoride fiber (H-filtration Membrane engineering Co., Ltd.), dopamine hydrochloride (sigma), polyethyleneimine (sigma), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, sigma), N-hydroxysuccinimide (NHS, sigma), ethanolamine hydrochloride (ETA, sigma)

Examples the apparatus used: scanning electron microscopy (SEM, Hitachi S4800), attenuated total reflectance infrared spectroscopy (ATR-IR, Bruker optics), fluorescence microscopy (Carl Zeiss, Thornwood, NY), AB Sciex 5600Q-TOF high resolution mass spectrometer (AB Sciex corporation), UHPLC system (Shimadzu), X-ray photoelectron spectroscopy (XPS), BIAcore T200 system (GE healthcare).

Example 1 preparation and characterization of polyvinylidene fluoride @ Polydopamine @ polyethyleneimine fibers

A bundle of the fibers was sonicated for half an hour using 12mL of a 10mM Tris-hydrochloride buffer solution (pH 8.5) to obtain activated fibers for use. And (3) taking the activated fiber, adding 80mg of dopamine, and stirring the solution overnight to obtain the polydopamine-coated fiber. The polydopamine-coated fibers were washed 3 times with ultrapure water and then immersed in 12mL of polyethyleneimine (20 mg/mL). Stirring the solution for 2 hours to obtain polyethyleneimine covered fibers; the fiber covered with the polyethyleneimine was immersed in 12mL of a xanthine oxidase solution (1mg/mL), and the solution was stirred for 3 hours to obtain a xanthine oxidase-covered fiber.

50, 100 and 200 activated fibers were taken, respectively, added with dopamine buffer, gently stirred for a period of time, 20 μ L was sampled at different time points, and the protein content in the solution was determined by the Bradford method. Quantification of fiber surface-bound proteins: the optimal ratio of the xanthine oxidase binding efficacy to the constant amount of xanthine oxidase was determined by the amount of fiber. The results show that: when 50 fibers are kept constant, the immobilized protein amount is maximum in each case after 3h incubation.

And (3) performing property characterization on the blank polyvinylidene fluoride fiber and the polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fiber by using a scanning electron microscope, infrared rays, X-ray spectral diffraction and a fluorescence microscope. Blank polyvinylidene fluoride fibers and polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers were incubated with anti-xanthine oxidase antibodies for 24h, respectively. Then washed 3 times with PBS buffer. Secondary antibodies were then added and incubated with blank polyvinylidene fluoride fibers and polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers for 2 hours. The fibers were then washed 3 times with T-PBS and photographed with a fluorescence microscope. Optical microscopy and immunofluorescence analysis of polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers show that: the surface of the modified polyvinylidene fluoride fiber is uniformly covered with xanthine oxidase.

And (3) infrared spectrum display: 1637cm^-1，3389cm^-1Matching with C ═ C, N-H of surface indole group after polydopamine treatment; 1184cm^-1，1068cm^-1，1642cm^-1Corresponding to non-aromatic C-N vibration and N-H vibration after coating polyethyleneimine; 1624cm^-1，1415cm^-1，1333cm^-1The absorption band of (b) was consistent with the characteristic absorption band of xanthine oxidase, indicating that the surface of the bonded fiber was covered with xanthine oxidase.

The results show that the polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fiber prepared by the method meets the requirements.

Example 2 Effect factor review of anti-gout drug screening method based on modified fiber enrichment

(1) Uses isoacteoside as model drug to optimize the modified elution solvent

Taking 10 polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers prepared in the example 1, adding 1.0mL (1.0mg/mL) of isoacteoside reference substance solution and 12mL of PBS buffer solution (pH6.7-6.9), mixing, incubating for 20min at 35 ℃, after incubation is completed, washing for 3 times by using 12mL of PBS buffer solution, carrying out liquid phase analysis on eluent, finally carrying out denaturation elution respectively by using 2mL of 10%, 30%, 50%, 70%, 90% and 100% acetonitrile-water solution (v/v) in volume concentration and 10%, 30%, 50%, 70% and 100% methanol-water solution (v/v) in volume concentration, and carrying out liquid phase analysis on the denaturation eluent respectively.

(2) Uses isoacteoside as model drug to optimize incubation time

The 10 polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers prepared in the above example 1 were taken, 1.0mL (1.0mg/mL) of isoacteoside control solution and 12mL PBS buffer (ph6.7-6.9) were added and mixed, incubation was performed at 35 ℃, incubation was performed after 5, 10, 20, 30, and 45min, after the incubation was completed, the fibers were washed 3 times with 12mL PBS buffer, the polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers were transferred to a 2mL test tube, denatured washing was performed with 70% methanol-water solution (v/v), and the denatured eluate was taken for liquid phase analysis.

(3) Uses isoacteoside as model drug to optimize incubation temperature

The 10 polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers prepared in the above example 1 were taken, 1.0mL (1.0mg/mL) of an isoacteoside reference solution and 12mL of PBS buffer (ph6.7-6.9) were added and mixed, incubation was performed at 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ for 20min, after completion of the incubation, the fibers were washed 3 times with 12mL of PBS buffer, the polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers were transferred to a 2mL test tube, denatured and washed with 70% methanol-water solution (v/v), and the denatured and eluted solution was taken for liquid phase analysis.

(4) Uses isoacteoside as model drug to optimize incubation pH value

The 10 polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers prepared in the above example 1 were taken, 1.0mL (1.0mg/mL) of isoacteoside control solution and 12mL PBS buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0, respectively) were added and mixed, incubated at 35 ℃ for 20min, after incubation, the fibers were washed 3 times with 12mL PBS buffer, the polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers were transferred to a 2mL test tube, denatured and washed with 70% methanol-water solution (v/v), and the denatured and eluted solution was taken for liquid phase analysis.

(5) Uses isoacteoside as model drug to optimize incubation ionic strength

10 polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers prepared in example 1 were taken, mixed with 1.0mL (1.0mg/mL) of an isolobal cimicifugaside control solution and 12mL of a buffer (50 mM, 160mM, 270mM, 380mM, 490mM, and 600mM in ionic strength, respectively) having a pH of 6.7 to 6.9, incubated at 35 ℃ for 20min, and after completion of incubation, the fibers were washed with 12mL of PBS buffer 3 times, the polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers were transferred to a 2mL test tube, denatured and washed with 70% methanol-water (v/v), and the denatured eluate was subjected to liquid phase analysis.

(6) Screening results for various factors

The purpose of washing is to eliminate the interference generated by the non-specifically bound compound, the invention inspects different washing times through a large number of experiments, and the result shows that the non-specifically bound compound can be sufficiently eliminated by washing three times with the PBS buffer solution. The denatured eluent can wash off the thrombin specifically bound to the fibers, and the results show that the elution efficiency of the 70% methanol-water elution solvent (v/v) is superior to that of other elution solvents.

Since the non-covalent interaction of small molecules with xanthine oxidase is mainly an electrostatic interaction, ionic strength and pH are important influencing factors. The experimental results show that: the pH value of the solution is about 6.7-6.9, and the ionic strength of the PBS buffer is 333-337 mM. The length of the incubation time and the temperature are also important factors influencing the affinity degree, and the result shows that the affinity degree of the isolobal cimicifugaside and the xanthine oxidase is the highest when the isolobal cimicifugaside is incubated for 20min at 35 ℃.

Example 3 application of the screening System to screening of anti-gout ingredients in Plantago asiatica

(1) Weighing 1g of plantain powder, adding 50mL of distilled water, refluxing for 1h, filtering, centrifuging the filtrate at 13400rpm for 10min, and sucking the supernatant for later use. Taking 1.0mL of the supernatant of the plantain herb extract, inserting 10 polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers into the supernatant, supplementing 35 ℃ of PBS buffer solution (pH6.7-6.9,333-337 mM) to 12mL, uniformly mixing the solution on a shaker, and incubating for 20 min. Then pulling out polyvinylidene fluoride @ polydopamine @ polyethyleneimine @ xanthine oxidase fibers, washing and cleaning the fibers with 12mL incubation buffer solution for 3 times, performing denaturing elution by using 70% methanol aqueous solution, and performing liquid chromatography and mass spectrometry on the denatured eluent

The chromatographic conditions were set as follows: syncronis AQ-C₁₈Chromatography column (2.1mm × 100mm, 1.7 μm), sample size 1 μ L; the temperature of the column incubator is 35 ℃; the flow rate is 0.4 mL/min; mobile phase, water (solvent a) containing 0.1% formic acid and acetonitrile (solvent B). The gradient is as follows: 5% of B (0-2 min), 5-30% of B (2.0-8.0 min), 30-95% of B (8.0-13.0 min), and 95-100% of B (13.0-17.0 min).

Time-of-flight mass spectra were as follows: ion spray voltage, -4.5 kV; collision energy: -35 eV; atomizer gas (gas 1), 55 psi; declustering voltage, -60V; heated gas (gas 2), 55 psi; ion source temperature: 550 ℃; curtain air pressure: 35 psi. Full scan data acquisition was performed, scanning m/z 100 to 1500 in negative ion mode.

(2) Chemical information of 9 active compounds in plantain is obtained by a liquid chromatography-mass spectrometry technology, and IC of the 9 compounds is determined by an ELISA method₅₀Value, affinity value of the compound with xanthine oxidase obtained by surface plasmon resonance (pD2)The results are shown in Table 1. The xanthine oxidase inhibitory activity of 7 compounds, namely baicalin A, scutellarin, plantaginoside, isoplantaginoside, hispidulin-7-O-beta-D-glucuronide, luteolin-7-O-diglucuronide and phillyrin C, is found for the first time.

TABLE 1 inhibition of xanthine oxidase by monomeric compounds

The method for screening the anti-gout drugs based on the modified fiber separation has accurate and reliable results. Compared with the traditional natural medicine active ingredient screening method, the method has the advantages of higher screening efficiency, less consumption of organic solvent and recycling.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.