Magnetic bead immobilized snake venom phospholipase A2 as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine separation, and particularly relates to magnetic bead immobilized snake venom phospholipase A2 as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out a coupling reaction on ferroferric oxide nanoparticles and (3-aminopropyl)-triethoxysilane in an organic solvent, washing, and drying to obtain aminated magnetic beads; and activating the aminated magnetic beads with glutaraldehyde, incubating with a snake venom phospholipase A2 solution, and reacting to obtain the magnetic bead immobilized snake venom phospholipase A2. The magnetic bead immobilized snake venom phospholipase A2 prepared by the invention can be used for rapidly and accurately fishing the snake venom phospholipase A2 toxin antidote from a mixture system, and has a better application prospect in the field of snake venom antidote research.

Description

Technical Field

[0001] This application belongs to the technical field of drug separation, and specifically relates to a magnetic bead-immobilized snake venom phospholipase A 2 and its preparation method and application. Background Art

[0002] Snake venom phospholipase A 2 has a wide range of toxic effects, including neurotoxicity, muscle toxicity, cytotoxicity and hemotoxicity, can cause injuries such as kidney and liver poisoning and systemic bleeding, induce the release of inflammatory mediators, vasodilator and vasoconstrictor mediators, etc., and has a very close relationship with snakebite poisoning. At present, antivenom serum, as a specific drug for the treatment of snakebite poisoning, has many disadvantages such as few varieties, too strong specificity, difficult to preserve, many adverse reactions, and it is difficult to meet the clinical needs. Developing new antivenom drugs that are economically accessible is an urgent need for the efficient treatment of snakebite poisoning.

[0003] In recent years, the treatment of snakebite poisoning with small molecule inhibitors has attracted wide attention. Prior art research has found that the small molecule inhibitor varfaradil has good inhibitory effects on the toxicity of Agkistrodon acutus, Naja naja, Gloydius blomhoffii, and Bungarus multicinctus. It has also been found that phospholipase A 2 inhibitors have good inhibitory effects on the coagulation disorder toxicity caused by different snake venoms including Agkistrodon acutus, and phospholipase A 2 inhibitors can widely inhibit the toxicity of snake venom and also show good inhibitory effects in animal experiments. In addition, natural drugs contain a large number of small molecules. However, the specific antivenom active ingredients in natural drugs are not clear, and the components of natural drug extracts are complex. Directly screening active ingredients from the mixture system will face difficulties such as large separation difficulty and many interfering factors. Therefore, developing a method for accurately capturing snake venom phospholipase A 2 inhibitors from natural drugs is of great significance for the subsequent development of new antivenom drugs. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a magnetic bead-immobilized snake venom phospholipase A 2 and its preparation method and application, and specifically adopt the following technical solutions: In the first aspect, the present invention provides a preparation method of a magnetic bead-immobilized snake venom phospholipase A 2 comprising the following steps: Coupling reaction of iron oxide (Fe 3 O 4 ) nanoparticles with (3-aminopropyl)-triethoxysilane in a solvent, washing, and drying to obtain amino-functionalized magnetic beads; Activating the amino-functionalized magnetic beads with an aqueous glutaraldehyde solution and then reacting with snake venom phospholipase A 2Co-incubate with the solution and react at a pH of 7-9 to obtain the magnetic bead-immobilized snake venom phospholipase A 2 During this process, when the pH is 7-9, it is the optimal reaction condition for snake venom phospholipase A 2 , and its catalytic activity is the best.

[0005] As a further preferred embodiment, the concentration of the glutaraldehyde crosslinking agent is 3%-5%, and the activation time is 5h-7h. If the glutaraldehyde concentration is lower than this range, the amino sites on the surface of the magnetic beads cannot be fully activated, resulting in a reduction in the active groups available for coupling; if the glutaraldehyde concentration is too high, it will cause excessive crosslinking of the enzyme, and the enzyme molecules will crosslink with each other and destroy the three-dimensional structure of the enzyme molecules, resulting in a decrease in the enzyme loading, and at the same time affecting the binding of the enzyme to the substrate and causing a decrease in the enzyme activity.

[0006] As a further preferred embodiment, the dosage ratio of the snake venom phospholipase A 2 solution to the amino-functionalized magnetic beads is 1mL:10mg; the concentration of the snake venom phospholipase A 2 solution is 100μg / mL-200μg / mL. If the enzyme concentration is too high, due to the limited binding sites on the magnetic beads, too much enzyme will cause the binding sites of the magnetic beads to reach saturation and continue to accumulate on the surface of the magnetic beads, which is not conducive to the exposure of the active sites, resulting in a decrease in the enzyme activity; if the enzyme concentration is too low, the enzyme loading is too low, and the enzyme molecules cannot be fully activated, which is not conducive to subsequent operations.

[0007] As a further preferred embodiment, the mass ratio of the iron oxide nanoparticles to (3-aminopropyl)-triethoxysilane is 1:2.5-4; among them, if (3-aminopropyl)-triethoxysilane is excessive, it will cause the silane layer on the surface of the magnetic beads to be too thick, and it is easy to fall off during subsequent reactions; if (3-aminopropyl)-triethoxysilane is too little, it will cause the amino group to not fully modify the magnetic beads, resulting in insufficient purity of the amino-functionalized magnetic beads and reducing the material utilization rate.

[0008] As a further preferred embodiment, the solvent is one or more of ethanol or water.

[0009] In a second aspect, the present invention provides a magnetic bead-immobilized snake venom phospholipase A 2 , which is prepared by the above preparation method.

[0010] In a third aspect, the present invention provides the above magnetic bead-immobilized snake venom phospholipase A 2 for use in screening for phospholipase A 2 inhibitors in natural drugs.

[0011] As a further preferred embodiment, the natural drugs include one or more of Paris polyphylla, Rohdea japonica, Hedyotis diffusa, Bidens pilosa, and Cassia occidentalis.

[0012] Fourthly, the present invention provides a method for screening phospholipase A 2 inhibitors from natural medicines, comprising the following steps: Mix the above-mentioned magnetic bead-immobilized snake venom phospholipase A 2 with the natural medicine extract, and after incubation, perform magnetic separation, washing, and elution with an organic solvent in sequence, collect the final eluate, and obtain the phospholipase A 2 inhibitor.

[0013] As a further preferred embodiment, the obtained eluate is detected by HPLC-MS / MS to obtain a fingerprint map, and the molecular structure of the snake venom phospholipase A 2 inhibitor is determined according to each characteristic peak in the map. After subsequent experimental verification, the phospholipase A 2 inhibitor components are determined, that is, the phospholipase A 2 inhibitor is obtained.

[0014] Fifthly, the present invention provides a phospholipase A 2 inhibitor screened by the above method.

[0015] The present invention provides the phospholipase A 2 inhibitor screened as above, which lays a foundation for the development of new anti-snake venom drugs.

[0016] The beneficial effects of the present invention are as follows: (1) The present invention uses Fe 3 O 4 magnetic beads as a carrier to achieve the immobilization of phospholipase A from cobra venom, enhancing the stability and reusability of the enzyme. 2

[0017] (2) The present invention combines the immobilized snake venom phospholipase A 2 with HPLC-MS / MS coupling technology to create a highly specific and sensitive screening platform for identifying and determining snake venom phospholipase A 2 inhibitors from complex samples such as natural medicine extracts.

[0018] (3) The magnetic bead-immobilized snake venom phospholipase A 2 adopted by the present invention can be directly used in the enzyme reaction system, eliminating the cumbersome purification process of traditional chromatography methods (such as ion exchange and gel filtration), and reducing the time and economic costs.

[0019] (4) The method of the present invention is environmentally friendly. The magnetic bead separation technology does not require centrifugation or filtration, reducing the generation of chemical waste and meeting the requirements of green chemistry.

[0020] (5) The present invention can quickly and accurately fish out cobra phospholipase A from the mixture system2 Toxin inhibitors have good application prospects in the field of snake venom antidote research.

[0021] (6) This invention explored the inhibitory effects of natural medicine extracts in nature on snake venom phospholipase A 2 and provided new ideas for the research and development of subsequent new anti-snake venom drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 Shown is a schematic flow diagram of fishing for active small molecules in the extract of Hedyotis diffusa Willd. with immobilized snake venom phospholipase A 2 ; Figure 2 Shown is the infrared spectrogram of the magnetic bead material; Figure 3 Shown is the transmission electron micrograph of magnetic bead-immobilized snake venom phospholipase A 2 ; Figure 4 Shown is the liquid chromatography diagram of the fishing result of the immobilized enzyme in the solution of rosmarinic acid single component; Figure 5 Shown is the liquid chromatography diagram of the fishing result of the immobilized enzyme in the mixed solution composed of marimastat and rosmarinic acid; Figure 6 Shown is the liquid chromatography diagram of the fishing result of the immobilized enzyme in the extract of Hedyotis diffusa Willd.; Figure 7 Shown is the total ion current mass spectrometry diagram of the fishing result of the immobilized enzyme in the extract of Hedyotis diffusa Willd.; Figure 8 Shown is the total ion current mass spectrometry diagram of the fishing result of the immobilized enzyme in the extract of Paris polyphylla Smith; Figure 9 Shown is the molecular docking simulation result of chlorogenic acid, o-coumaric acid and caffeic acid with snake venom phospholipase A 2 ; Figure 10 Shown is the molecular docking simulation result of rutin, kaempferol and luteoloside with snake venom phospholipase A 2 ; Figure 11 Shown is the first-order and second-order mass spectrometry diagrams of caffeic acid. DETAILED DESCRIPTION OF THE INVENTION

[0024] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are partial embodiments of the present application, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0025] Example 1 A preparation method of magnetic bead-immobilized snake venom phospholipase A 2 comprises the following steps: (1) Synthesize Fe 3 O 4 nanoparticles by coprecipitation method: Take 8.1 g of FeCl 3 ·6H 2 O and 6.2 g of FeSO 4 ·7H 2 O in a 250 mL three-necked flask, add 150 mL of pure water to dissolve, under the conditions of nitrogen protection and 70 °C for the mixed solution, add 18 mL of concentrated ammonia water, continue to stir for 1 hour after mixing, and the product Fe 3 O 4 nanoparticles are washed alternately with 500 mL of water and ethanol, and stored sealed after freeze-drying; (2) Prepare amino-functionalized magnetic beads: Take 1 g of Fe 3 O 4 nanoparticles and 3 g of (3-aminopropyl)-triethoxysilane in a 150 mL conical flask, add 60 mL of ethanol, and carry out a coupling reaction by stirring at 60 °C for 24 hours. After washing with 300 mL of water and ethanol and drying, amino-functionalized magnetic beads are obtained; (3) Take 10 mg of the amino-functionalized magnetic beads obtained in step (2) and activate them with 10 mL of 4% glutaraldehyde aqueous solution for 6 hours. After alternately washing with 20 mL of ethanol and water, mix with 1 mL of 100 μg / mL snake venom phospholipase A 2 solution (adjust the pH to 7.9 with PBS solution), react overnight at 4 °C to obtain magnetic bead-supported immobilized snake venom phospholipase A 2 , wash with 3 mL of PBS buffer solution and measure its enzyme loading and enzyme activity. The immobilized enzyme loading is 6.5 μg / mg, and the immobilized enzyme activity is 89% of the free enzyme.

[0026] Figure 2 is the infrared spectrogram of the material preparation process. Among them, the Fe-O characteristic peak of Fe 3 O 4 is between 570 cm -1 -580 cm -1 ; in the amino-functionalized magnetic beads, the bending vibration peak of Si-O is at 1020 cm -1Nearby, 1370 cm -1 is the characteristic absorption peak of the -C-N- bond and belongs to the functional group valence bond introduced by (3-aminopropyl)-triethoxysilane. 1580 cm -1 is the N-H deformation vibration peak, and the characteristic peaks at 2930 cm -1 and 2850 cm -1 are the symmetric and asymmetric stretching vibrations of -CH 2 - in (3-aminopropyl)-triethoxysilane. In the infrared spectrogram of glutaraldehyde-activated amino-functionalized magnetic beads, the stretching vibration peak at 1630 cm -1 is for -C=N-, and the above results can prove the successful synthesis of the material.

[0027] Figure 3 Shown in the figure is the transmission electron microscope image of magnetic bead-immobilized snake venom phospholipase A 2 ; it can be seen that in the transmission electron microscope image of the material, the magnetic beads are dispersed spheres, and phospholipase A 2 is uniformly wrapped around the surface of the magnetic beads.

[0028] Example 2 A preparation method of magnetic bead-immobilized snake venom phospholipase A 2 comprises the following steps: (1) Synthesize Fe 3 O 4 nanoparticles by the co-precipitation method: Take 8.1 g of FeCl 3 ·6H 2 O and 6.2 g of FeSO 4 ·7H 2 O in a 250 mL three-necked flask, add 150 mL of pure water to dissolve, and under the conditions of nitrogen protection and 70 °C, add 18 mL of concentrated ammonia water. After mixing, continue stirring for 1 hour. The product Fe 3 O 4 nanoparticles are washed alternately with 500 mL of water and ethanol, and stored sealed after freeze-drying; (2) Prepare amino-functionalized magnetic beads: Take 1 g of Fe 3 O 4 nanoparticles and 3 g of (3-aminopropyl)-triethoxysilane in a 150 mL conical flask, add 60 mL of ethanol, and carry out a coupling reaction by stirring at 60 °C for 24 hours. After washing with 300 mL of water and ethanol, amino-functionalized magnetic beads are obtained after drying; (3) Take 10 mg of the amino-functionalized magnetic beads obtained in step (2) and activate them with 10 mL of 5% glutaraldehyde aqueous solution for 6 hours. After washing alternately with 20 mL of ethanol and water, they are reacted with 1 mL of 100 μg / mL snake venom phospholipase A 2Mix with a solution (adjust the pH to 7.9 using PBS solution), react overnight at 4°C to obtain immobilized snake venom phospholipase A with magnetic beads as the carrier. 2 Wash with 3 mL of PBS buffer and measure its enzyme loading and enzyme activity. The immobilized enzyme loading is 4.2 μg / mg, and the immobilized enzyme activity is 80% of the free enzyme.

[0029] Example 3 A preparation method of magnetic bead-immobilized snake venom phospholipase A 2 comprises the following steps: (1) Synthesize Fe 3 O 4 nanoparticles: Take 8.1 g of FeCl 3 ·6H 2 O and 6.2 g of FeSO 4 ·7H 2 O and place them in a 250 mL three-necked flask. Add 150 mL of pure water to dissolve. Under the protection of nitrogen and at 70°C, add 18 mL of concentrated ammonia water. After mixing, continue stirring for 1 hour. The product Fe 3 O 4 nanoparticles are washed alternately with 500 mL of water and ethanol, and stored sealed after freeze-drying; (2) Prepare amino-functionalized magnetic beads: Take 1 g of Fe 3 O 4 nanoparticles and 3 g of (3-aminopropyl)-triethoxysilane in a 150 mL conical flask. Add 60 mL of ethanol and stir at 60°C for 24 hours for the coupling reaction. Wash with 300 mL of water and ethanol, and dry to obtain amino-functionalized magnetic beads; (3) Take 10 mg of the amino-functionalized magnetic beads obtained in step (2) and activate them with 10 mL of 4% glutaraldehyde aqueous solution for 7 hours. Wash alternately with 20 mL of ethanol and water, and mix with 1 mL of 100 μg / mL snake venom phospholipase A 2 solution (adjust the pH to 7.9 using PBS solution). React overnight at 4°C to obtain immobilized snake venom phospholipase A with magnetic beads as the carrier 2 Wash with 3 mL of PBS buffer and measure its enzyme loading and enzyme activity. The immobilized enzyme loading is 6.2 μg / mg, and the immobilized enzyme activity is 67% of the free enzyme.

[0030] Example 4 A preparation method of magnetic bead-immobilized snake venom phospholipase A 2 comprises the following steps: (1) Synthesize Fe 3 O 4 nanoparticles: Take 8.1 g of FeCl 3 ·6H 2O and 6.2 g of FeSO 4 ·7H 2 O were added to a 250 mL three-necked flask, and 150 mL of pure water was added for dissolution. Under the protection of nitrogen and at 70 °C, 18 mL of concentrated ammonia water was added to the mixed solution. After mixing, stirring was continued for 1 hour. The product Fe 3 O 4 nanoparticles were washed alternately with 500 mL of water and ethanol, and stored sealed after freeze-drying; (2) Preparation of amino-functionalized magnetic beads: 1 g of Fe 3 O 4 nanoparticles and 3 g of (3-aminopropyl)-triethoxysilane were placed in a 150 mL conical flask, 60 mL of ethanol was added, and the coupling reaction was carried out under stirring at 60 °C for 24 hours. After washing with 300 mL of water and ethanol and drying, amino-functionalized magnetic beads were obtained; (3) 10 mg of the amino-functionalized magnetic beads obtained in step (2) were activated with 10 mL of 4% glutaraldehyde aqueous solution for 6 hours, washed alternately with 20 mL of ethanol and water, and mixed with 1 mL of 200 μg / mL snake venom phospholipase A 2 solution (adjusted to pH 7.9 with PBS solution), and reacted overnight at 4 °C to obtain immobilized snake venom phospholipase A 2 supported on magnetic beads. It was washed with 3 mL of PBS buffer solution and its enzyme loading and enzyme activity were measured. The immobilized enzyme loading was 14 μg / mg, and the immobilized enzyme activity was 78% of the free enzyme.

[0031] Example 5 Explore the fishing ability of the immobilized snake venom phospholipase A 2 in a single-component solution, as follows: Rosmarinic acid (a positive inhibitor of snake venom phospholipase A 2 ) was dissolved in PBS buffer solution to prepare a 3 mg / mL solution, named S0. 200 μL of the solution was incubated with 10 mg of magnetic bead-immobilized phospholipase A 2 at room temperature for 30 minutes, followed by magnetic separation. The supernatant was taken out and named S1; 200 μL of PBS solution was used to wash the magnetic beads twice, and the eluates were named W1 and W2 in sequence; the magnetic beads were eluted twice with 200 μL of 25% (v / v) acetonitrile aqueous solution, and the supernatant of each elution was collected and named E1 and E2 in sequence; after filtering each batch of supernatant, high-performance liquid chromatography was used for analysis. The chromatographic column was Supersil ODS2 (2.1 mm × 100 mm, 2 μm particle size, 120 Å pore volume), the injection volume was 5 μL, the column temperature was 35 °C, and the detection wavelength was 220 nm.

[0032] The liquid phase elution conditions are as follows: Mobile phase A: 0.1% formic acid aqueous solution (v / v); Mobile phase B: acetonitrile. The total flow rate is 0.5 mL / min, and isocratic elution is carried out at a ratio of 55% mobile phase A: 45% mobile phase B for 15 minutes.

[0033] The results are as Figure 4 shown. The retention time of the rosmarinic acid chromatographic peak is 2.9 minutes - 3.3 minutes. After separating the magnetic beads from S0, the magnetic beads are rinsed with PBS solution to remove the non-specifically adsorbed rosmarinic acid on the surface; there is no longer a characteristic peak of rosmarinic acid in W2, indicating that the non-specifically adsorbed rosmarinic acid has been completely rinsed off; however, rosmarinic acid is still detected in the eluate (E1, E2), indicating that the magnetic beads have specific adsorption for rosmarinic acid and can be eluted; the above results show that the magnetic bead material has a certain fishing ability for rosmarinic acid.

[0034] Example 6 Explore the fishing ability of the immobilized snake venom phospholipase A in Example 1 2 in the mixed system, specifically as follows: Prepare a mixture model S0 by dissolving rosmarinic acid and marimastat in PBS buffer; take 200 μL of the mixed solution and incubate it with 10 mg of immobilized phospholipase A 2 for 30 minutes. After magnetic separation, take out the supernatant and name it S1; take 200 μl of PBS solution to rinse the magnetic beads twice, and name the eluates W1 and W2 in sequence; elute the magnetic beads twice with 200 μl of 25% (v / v) acetonitrile aqueous solution, collect the supernatant each time, and name it E1 and E2 in sequence; filter each batch of supernatant and analyze it by high performance liquid chromatography. The chromatographic column is Supersil ODS2 (2.1 mm × 100 mm, 2 μm particle size, 120 Å pore volume), the injection volume: 5 μL, the column temperature is 35 °C, and the detection wavelength is 220 nm.

[0035] The liquid phase elution conditions are as follows: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile. The total flow rate is 0.5 mL / min; Gradient elution is carried out for 20 minutes, and the elution gradient is shown in Table 1: Table 1 The results of its liquid chromatography detection are as Figure 5As shown, it can be seen from the figure that the retention time of the liquid chromatography peak of marimastat is 3.4 minutes - 3.9 minutes, and the retention time of the liquid chromatography peak of rosmarinic acid is 9.5 minutes - 9.8 minutes. Marimastat has been completely removed in the elution (W1, W2) part and is almost absent in the eluate (E1, E2), indicating that marimastat does not specifically bind to the magnetic beads. The content of rosmarinic acid in the eluate (E1, E2) is much higher than that in the eluent (W1, W2). The elution process mainly removes the non-specifically adsorbed rosmarinic acid on the surface of the magnetic beads, and the eluate releases the specifically bound rosmarinic acid. The chromatogram results of each component show that the immobilized snake venom phospholipase A 2 has almost no specific binding with the negative inhibitor marimastat in the mixed system, but shows good specific adsorption to rosmarinic acid. The above results indicate that the prepared immobilized snake venom phospholipase A 2 has good selective recognition ability for the inhibitor rosmarinic acid in the mixed solution and has good application potential in the fishing of phospholipase A 2 inhibitors.

[0036] Example 7 Explore the fishing ability of the immobilized snake venom phospholipase A 2 in the extract of Hedyotis diffusa (the process schematic diagram is as Figure 1 shown) The extract paste of Hedyotis diffusa was obtained by ultrasonic extraction method. After being configured into a solution (S), 200 μL of the mixed solution was taken and incubated with 10 mg of immobilized phospholipase A 2 for 30 minutes; the magnetic beads were washed with 200 μL of PBS solution, and the eluent was named W. Then, the magnetic beads were continuously eluted with 200 μL of 25% (v / v) acetonitrile aqueous solution, and the supernatant was collected and named E; each batch of supernatant was filtered and analyzed by high performance liquid chromatography - tandem mass spectrometry (HPLC - MS / MS).

[0037] Liquid phase conditions: The chromatographic column is Supersil ODS2 (2.1 mm × 100 mm; 2 μm particle size, 120 Å pore volume), mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; the total flow rate is 0.3 mL / min; the injection volume: 5 μL; the column temperature is 35 °C, and the detection wavelength: 254 nm.

[0038] Gradient elution was carried out for 40 minutes, and the elution gradient is shown in Table 2: Table 2 Tandem mass spectrometry conditions: Collision gas: nitrogen; Ion source type: electrospray ionization source; Spray gas: 50 psi; Auxiliary heating gas: 50 psi; Ion spray voltage: -4500 V; Ion source temperature: 550 °C; Gas flow rate: 8.0 L / min. Detection was carried out in the negative ion mode. The mass range of the first-stage mass spectrometry scan was: m / z 100 - 1000, the collision energy was -10 eV, and the voltage applied in the ion source was -80 eV. The mass range of the second-stage mass spectrometry scan was: m / z 50 - 1000, the collision energy was -35 eV ± 15 eV, and the voltage applied in the ion source was -80 eV. The results are as Figure 6 shown. It can be seen from the figure that there are many substances other than phospholipase A 2 inhibitors in the Hedyotis diffusa sample. After removing the non-specifically adsorbed substances with the eluent, a large number of small molecules specifically adsorbed with phospholipase A 2 were detected in the eluate, and they are the target compounds. To further identify the target compounds in Hedyotis diffusa, combined with mass spectrometry analysis, the total ion chromatograms of the sample and the eluate are as Figure 7 shown; it can be known from Figure 7 that active small molecules were detected in the eluate. By analyzing the mass spectrometry fragmentation structure, consulting the literature and comparing with the mass spectrometry database to further analyze the chemical structure of the small molecules, the following three natural medicine active small molecules were obtained.

[0039] The chemical structures of the active substances in Hedyotis diffusa were screened out as follows: Example 8 Explore the monomer inhibition effect of the natural small molecule inhibitor obtained in Example 7 on snake venom phospholipase A 2 According to the identification results of HPLC-MS / MS, three active small molecules, caffeic acid, o-coumaric acid, and chlorogenic acid, were detected in the eluate. Therefore, these three compounds were used to prepare solutions with a concentration of 2 mg / L in PBS solution and incubated with immobilized snake venom phospholipase A 2 for 30 minutes. In the control group, the immobilized phospholipase A 2 was directly placed in PBS solution and incubated for 30 minutes. The changes in enzyme activity before and after incubation of each group were measured.

[0040] Enzyme activity assay method: Using cresol red as an indicator, add lecithin (0.875 mmol / L), potassium chloride (100 mmol / L), sodium chloride (100 mmol / L), calcium chloride (10 mmol / L), and cresol red (0.02 mg / mL) to 50 mL of Tris buffer solution (1 mmol / L). The above concentrations are all the final concentrations of the substances in the solution; Phospholipase A 2By breaking down lecithin to produce fatty acids, the pH changes. Cresol red changes color with the pH change, which can be used to detect the extent of the enzyme-catalyzed reaction. 2 After 5 minutes of reaction, the absorbance before and after the reaction was read at 572 nm using a multifunctional microplate reader, and the immobilized phospholipase A was calculated based on the difference. 2 Activity. The results are shown in Table 3. The three compounds have an activity against snake venom phospholipase A 2 All of them had a certain inhibitory effect. The inhibition rates of caffeic acid, o-coumaric acid, and chlorogenic acid were 47.60%, 56.45%, and 28.48%, respectively. The inhibition experiment results verified that the immobilized snake venom phospholipase A 2 Effectiveness and practicality in drug screening.

[0041] Table 3 Example 9 In order to further verify the immobilized snake venom phospholipase A 2 The effectiveness of drug screening was investigated by combining the natural drug small molecule obtained in Example 7 with snake venom phospholipase A 2 Perform molecular docking simulation, which is specifically implemented as follows: Snake venom phospholipase A 2 The three-dimensional structure of was downloaded from the PDB (https: / / www.rcsb.org / ) database (PDBID: 2OSH), and the structures of chlorogenic acid, o-coumaric acid, and caffeic acid were downloaded from the PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) database. Pretreatment was performed in AutoDock Tools (4.2.6) to remove water molecules, and molecular docking was performed using AutoDock Vina (1.2.5) software, and the molecular docking results were visualized using Pymol (3.1.3).

[0042] Chlorogenic acid, o-coumaric acid, caffeic acid and phospholipase A 2 The docking results are as follows Figure 9 As shown, chlorogenic acid is Figure 9 As shown in A, o-coumaric acid as Figure 9 As shown in B, caffeic acid Figure 9 As shown in C; As can be seen from the figure, all three of them are connected to phospholipase A through hydrogen bonds 2 The binding sites are the catalytic residue Tyr 28, the characteristic active residue Asp 49, the characteristic active sites His 48 and Asp 49, and the binding energies are -7.3 kcal / mol, -7.2 kcal / mol, and -7.0 kcal / mol, respectively.

[0043] His 48 and Asp 49 are phospholipase A 2 The important characteristic active site of phospholipase A plays a key role in the enzymatic hydrolysis catalysis reaction; Tyr 28 2 Ca2+ 2+ It plays a key role in the process and can effectively promote phospholipase A 2 The catalytic activity of chlorogenic acid, o-coumaric acid, and caffeic acid interact with phospholipase A through these three important residues. 2 Binding causes the spatial configuration of the active site to change, thereby affecting the catalytic ability of the enzyme, which is the inhibitory factor of phospholipase A 2 Therefore, molecular docking simulation not only supports and verifies the inhibitory effect of natural drug active small molecules, but also further clarifies the mechanism of its inhibitory enzyme activity.

[0044] Example 10 In order to further verify the immobilized snake venom phospholipase A 2 Screening applicability of immobilized snake venom phospholipase A in natural drug extracts 2 Application in the Paris polyphylla extract to fish out inhibitory small molecules.

[0045] The extract paste of Paris polyphylla was obtained by ultrasound-assisted method and prepared into solution (S). 200 µL of the solution was mixed with 10 mg of immobilized phospholipase A. 2 Incubate for 30 minutes; wash the magnetic beads with 200 µL PBS solution, the eluent is named W, and then elute the magnetic beads with 200 µL 25% (v / v) acetonitrile aqueous solution, collect the supernatant, named E; filter each batch of supernatant and analyze it using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0046] Liquid phase conditions: chromatographic column: Supersil ODS2 (2.1 mm × 100mm; 2μm particle size, 120Å pore volume), injection volume: 5μL; column temperature: 35℃, detection wavelength: 203nm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; total flow rate: 0.3mL / min.

[0047] Gradient elution was performed for 21 minutes. The elution gradient is shown in Table 4: Table 4 Tandem mass spectrometry conditions: collision gas: nitrogen; ion source type: electrospray ionization source; spray gas: 50psi; auxiliary heating gas: 50psi; ion spray voltage: -4500V; ion source temperature: 550℃; gas flow rate: 8.0L / min. Detection in negative ion mode, the mass range of the primary mass spectrometry scan is: m / z 100-1000, the collision energy is -10eV, and the voltage applied to the ion source is -80eV. The mass range of the secondary mass spectrometry scan is: m / z 50-1000, the collision energy is -35eV±15eV, and the voltage applied to the ion source is -80eV.

[0048] Combined with mass spectrometry analysis, the total ion current of the sample and eluent is as follows Figure 8 As shown in the figure, there are many phospholipase A in Paris polyphylla samples. 2 Substances other than inhibitors were detected in the eluate after nonspecific adsorption was removed. 2 The small molecules specifically adsorbed are the target compounds. The target compounds in Paris polyphylla were further identified, and the chemical structures of the active substances in Paris polyphylla were screened out as follows: Embodiment 11 Study on the effect of the natural small molecule inhibitor obtained in Example 10 on snake venom phospholipase A 2 Monomer inhibitory effect.

[0049] According to the results of HPLC-MS / MS, four active small molecules, namely, luteolin, o-coumaric acid, rutin, and kaempferol, were detected in the eluate. Therefore, these four compounds were selected and prepared into a solution with a concentration of 2 mg / L using PBS solution and then reacted with immobilized snake venom phospholipase A. 2 After incubation for 30 minutes, the control group was immobilized with snake venom phospholipase A 2 The cells were directly placed in PBS solution and incubated for 30 minutes, and the changes in enzyme activity before and after incubation were measured in each group.

[0050] Enzyme activity determination method: using cresol red as an indicator, add lecithin (0.875mmol / L), potassium chloride (100mmol / L), sodium chloride (100mmol / L), calcium chloride (10mmol / L), cresol red (0.02mg / mL) to 50mL Tris buffer solution (1mmol / L). The above concentrations are the final concentrations of the substances in the solution; phospholipase A 2 By breaking down lecithin to produce fatty acids, the pH changes. Cresol red changes color with the pH change, which can be used to detect the extent of the enzyme-catalyzed reaction. 2After 5 minutes of reaction, the absorbance before and after the reaction was read at 572 nm using a multifunctional microplate reader, and the immobilized phospholipase A was calculated based on the difference. 2 Activity. The results are shown in Table 5. The four compounds have an activity against snake venom phospholipase A 2 All of them had a certain inhibitory effect. The inhibition rates of luteolin, o-coumaric acid, rutin, and kaempferol were 42.92%, 56.45%, 28.01%, and 44.44%, respectively. The results of the inhibition experiment verified that the immobilized snake venom phospholipase A 2 Effectiveness and practicality in drug screening.

[0051] Table 5 Example 12 In order to further verify the immobilized snake venom phospholipase A 2 In terms of the effectiveness of drug screening, the natural drug small molecule obtained in Example 10 was combined with snake venom phospholipase A 2 Perform molecular docking simulation, which is specifically implemented as follows: Snake venom phospholipase A 2 The three-dimensional structure of was downloaded from the PDB (https: / / www.rcsb.org / ) database (PDBID: 2OSH), and the structures of rutin, kaempferol, and luteolin were downloaded from the PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) database. Molecular pretreatment was performed by dehydration in AutoDock Tools (4.2.6), and molecular docking was performed using AutoDock Vina (1.2.5) software, and the molecular docking results were visualized using Pymol (3.1.3).

[0052] Rutin, kaempferol, luteolin and phospholipase A 2 The docking results are as follows Figure 10 As shown, rutin Figure 10 As shown in A, kaempferol as Figure 10 As shown in B, luteolin as Figure 10 As shown in C; As can be seen from the figure, all three of them are connected to phospholipase A through hydrogen bonds 2 The binding site of rutin is the catalytic residue Tyr 28, and the binding sites of kaempferol and luteolin are both the characteristic active residue His 48, with binding energies of -5.6 kcal / mol, -8.7 kcal / mol, and -7.0 kcal / mol, respectively.

[0053] His 48 is phospholipase A 2 It is one of the important residues of the catalytic dyad, plays a key role in the enzymatic hydrolysis catalysis reaction, and is an important characteristic active site of the enzyme; Tyr 28 in phospholipase A2 Combined with Ca 2+ plays a key role in the process and can therefore effectively promote phospholipase A 2 catalysis. Rutin, kaempferol, and luteoloside are all bound to important residues through hydrogen bonding, resulting in a change in the spatial configuration of the active site and thus affecting the catalytic ability of the enzyme, which is the reason for inhibiting phospholipase A 2 activity. It can be seen from this that through molecular docking simulation, not only the inhibitory effect of natural small molecules is supported and verified, but also the mechanism of its inhibition of enzyme activity is further clarified.

[0054] Example 13 Identify the structure of the small molecule compound fished out in Example 7 Taking caffeic acid as an example, the eluate was analyzed by HPLC-MS / MS technology. The first-order and second-order fragments of the target compound are as Figure 11 shown, and the structure of the compound was analyzed: 1) Mass spectrometry characteristics: The actually measured mass-to-charge ratio of this substance is 179.0355. By consulting the literature and comparing the mass spectrometry database, the mass-to-charge ratio of caffeic acid is 179.0352. The deviation between the two is within the error tolerance range (<5 ppm), and this substance can be identified as caffeic acid.

[0055] 2) Retention time: The retention time of this compound is 9.39 min, and the retention time of caffeic acid is 9.38 min, with a difference of 0.01 min, indicating that the actual retention time of the compound is highly consistent with the retention time of caffeic acid, thus further verifying that this substance is caffeic acid.

[0056] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples have been used in this article to elaborate on the principles and implementation manners of the present application. The descriptions of the above embodiments are only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are only illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all belong to the protection scope of the present application.

Claims

1. A method for preparing magnetic bead-immobilized snake venom phospholipase A2, characterized in that: The following steps are involved: The ferrosoferric oxide nanoparticles are coupled with (3-aminopropyl)-triethoxysilane in a solvent, washed, and dried to obtain amino magnetic beads; The amino magnetic beads are activated by glutaraldehyde aqueous solution and then incubated with a snake venom phospholipase A2 solution, and reacted at a pH of 7-9 to obtain the magnetic bead-immobilized snake venom phospholipase A2.

2. The preparation method according to claim 1, characterized in that: The mass concentration of the glutaraldehyde aqueous solution is 3%-5%, and the activation time is 5h-7h.

3. The preparation method according to claim 1, characterized in that: The usage ratio of the snake venom phospholipase A2 solution and the amino-modified magnetic beads is 1 mL:10 mg; the concentration of the snake venom phospholipase A2 solution is 100 μg / mL-200 μg / mL.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the ferrosoferric oxide nanoparticles to (3-aminopropyl)-triethoxysilane is 1:2.5-4; and the solvent is one or more of ethanol or water.

5. A magnetic bead-immobilized snake venom phospholipase A2, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the magnetic beads-immobilized snake venom phospholipase A2 according to claim 5 in screening phospholipase A2 inhibitors in natural medicines.

7. The use according to claim 6, characterized in that: The natural medicines include one or more of Paris polyphylla, Dieffenbachia glabra, Hedyotis diffusa, Bidens pilosa, and Psoralea corylifolia.

8. A method for screening phospholipase A2 inhibitors in natural medicines, characterized in that: The following steps are involved: The magnetic bead-immobilized snake venom phospholipase A2 of claim 5 is mixed with a natural drug extract, and after incubation, magnetic separation, elution, and organic solvent elution are performed in sequence, and the final eluate is collected to obtain the phospholipase A2 inhibitor.

9. The method according to claim 8, characterized in that The following steps are also included: The obtained eluate is detected by HPLC-MS / MS to obtain a fingerprint spectrum, and the molecular structure of the phospholipase A2 inhibitor is determined according to each characteristic peak in the spectrum, thus obtaining the phospholipase A2 inhibitor.

10. A phospholipase A2 inhibitor, characterized in that The method is screened by any one of claims 8 to 9.

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