Method for high-throughput screening of monoamine oxidase B inhibitor

Abstract

The invention belongs to the technical field of active substance screening, and particularly relates to a method for high-throughput screening of a monoamine oxidase B inhibitor. The method comprises the following steps: incubating monoamine oxidase B and a drug-containing brain homogenate extracting solution, removing components which are not specifically bound by using an affinity ultrafiltration technology, eluting by using methanol to obtain a potential ligand, and identifying by using UPLC-Q-Exactive-MS (Ultra Performance Liquid Chromatography-Quantitative Exactive-Mass Spectrometry). Meanwhile, inactivated monoamine oxidase B is used as a control, interference of the control, false positive and non-specific binding is eliminated, the in-vitro monoamine oxidase B inhibition effect of the identification ligand is measured, and the result proves that the compound screened by using the affinity ultrafiltration-liquid chromatography-mass spectrometry technology has the monoamine oxidase B inhibition activity. The method disclosed by the invention is rapid, simple, convenient, stable, high in precision and good in reproducibility, and has a wide application prospect in the aspect of rapidly screening target compounds in natural medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance screening, and particularly relates to a method for high-throughput screening of monoamine oxidase B inhibitors. Background Art

[0002] Parkinson's disease is a neurodegenerative disorder characterized by complex, multiple motor and non-motor signs and symptoms that typically affects individuals over the age of 60. The incidence and prevalence of Parkinson's disease are increasing rapidly worldwide as the population ages. The Global Neurological Disease Survey shows that Parkinson's disease may even be the fastest growing neurological disease worldwide. The most effective treatments for Parkinson's disease motor symptoms are those that modulate the dopamine system, either by providing exogenous dopamine (levodopa), enhancing dopamine receptor activity (dopamine receptor agonists), or interfering with dopamine catabolism (monoamine oxidase-B inhibitors). MAO-B inhibitors are safe, well tolerated, and provide symptomatic benefits from the early to late stages of the disease, either as monotherapy or in combination with levodopa. However, a potentially serious complication of taking high doses of MAO-B inhibitors is the "cheese effect," which is a cause for concern. Therefore, an efficient and rapid method is urgently needed to screen natural, highly effective, and low-toxic MAO-B inhibitors from the treasure trove of traditional Chinese medicine.

[0003] Studies have found that many Chinese medicines or their components show effective neuroprotective effects in in vitro and in vivo models of Parkinson's disease. However, the process of discovering bioactive ingredients from Chinese herbal medicines or formulas with complex chemical compositions is time-consuming and laborious, and there are also serious human errors. The efficiency of directly screening bioactive compounds from plants is very low. Therefore, it is necessary to find a method for efficiently and quickly screening MAO-B inhibitors to provide a basis for screening MAO-B plant-derived inhibitors. Summary of the invention

[0004] In view of the above problems, the present invention proposes a method for high-throughput screening of monoamine oxidase B inhibitors. The present invention utilizes affinity ultrafiltration-liquid chromatography-mass spectrometry technology to discover MAO-B inhibitors that can cross the blood-brain barrier from Chinese herbal medicines or formulas with complex chemical components. The operation is simple, reliable, and time-saving, and the method can more realistically simulate the brain environment under physiological conditions, which is convenient for promotion and application.

[0005] The present invention adopts the following technical solutions:

[0006] A method for high-throughput screening of monoamine oxidase B inhibitors comprises the following steps:

[0007] (1) homogenizing the drug-containing rat brain tissue, centrifuging once to precipitate protein, centrifuging twice, taking the supernatant and blowing it dry with nitrogen, and then dissolving the residue in PBS to obtain a drug-containing brain homogenate extract;

[0008] More specifically, 12 ml of drug-containing rat brain tissue was homogenized, then centrifuged at 3000 rpm for 10 min, and the supernatant was vortexed with three times the amount of acetonitrile for 5 min to precipitate protein; then centrifuged at 12000 rpm for 10 min, and the supernatant was dried. Finally, the resulting residue was dissolved in 400 μL PBS to obtain a drug-containing brain homogenate extract.

[0009] (2) incubating the drug-containing brain homogenate extract with monoamine oxidase B, and then ultrafiltering the mixture to obtain a conjugate;

[0010] More specifically, the drug-containing brain homogenate extract and MAO-B are evenly mixed and incubated at 37° C. for 60 minutes to allow MAO-B to combine with the active ingredients in the drug-containing brain homogenate extract.

[0011] The incubated mixture was transferred to a 30KD ultrafiltration centrifuge tube, centrifuged at 4°C and 12000 rpm for 15 min, and the filtrate was discarded; PBS was added to the upper layer of the ultrafiltration centrifuge tube, mixed, centrifuged at 4°C and 12000 rpm for 15 min, and the filtrate was discarded. Repeat the operation 3 times;

[0012] (3) adding a 70% methanol aqueous solution to the complex, dissociating for 10 min, centrifuging at 4° C. and 12,000 rpm for 15 min, repeating the operation three times, collecting the dissociation solution and drying it to obtain a dissociation solution residue;

[0013] (4) The residue of the dissociation solution was dissolved in a 50% by volume methanol aqueous solution, filtered through a microporous membrane, and analyzed and identified using UPLC-Q-Exactive-MS.

[0014] In the present invention, the volume ratio of drug-containing brain homogenate extract to monoamine oxidase B is 5:1.

[0015] The incubation time in step (2) is 1 h and the incubation temperature is 37°C.

[0016] In step (3), the incubation time is 10 min and the dissociation temperature is 37°C.

[0017] The liquid phase conditions for analysis and identification in step (4) are: Chromatographic column: ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm; Waters, USA); column temperature: 30°C; flow rate: 0.2 mL·min -1 ; Injection volume: 3 μL; Mobile phase: Phase A is acetonitrile; Phase B is water containing 0.1% formic acid; Gradient elution.

[0018] The gradient elution program was as follows: 0-8 min, 2%-12% A; 8-16 min, 12-16% A; 16-19 min, 16% A; 19-22 min, 16%-19% A; 22-25 min, 19% A; 25-30 min, 19%-25% A; 30-32 min, 25% A; 32-35 min, 25%-30% A; 35-38 min, 30%-40% A; 38-40 min, 40%-50% A; 40-45 min, 50%-90% A; 45.2-50 min, 2% A.

[0019] The mass spectrometry conditions for analysis and identification in step (4) are as follows: electrospray ionization source (ESI); negative ion mode; capillary voltage 3500 V; capillary temperature 350° C.; sheath gas N2: 45 units, auxiliary gas N2: 15 units; collision energy: 10, 30, 50 eV; detection method: Full MS / ddMS 2 (Full MS resolution is 70,000FWHM, dd-MS 2 Resolution: 17,500 FWHM); mass scan range: m / z 100~1500; S–Lens RF Level: 55.

[0020] The MAO-B inhibitory effects of the identified chemical components were determined to verify the results.

[0021] The process of obtaining drug-containing rat brain tissue is as follows:

[0022] (1) crushing the plant sample, adding 10 times the volume fraction of 70% ethanol aqueous solution, heating and refluxing extraction three times to obtain an extract, and vacuum rotary evaporation to obtain a plant extract;

[0023] (2) Male SD rats were given plant extracts at a dosage of 10 mL / kg / d. Three days after intragastric administration, the drug-containing rat brain tissues were obtained.

[0024] The plant is Cistanche deserticola, and the plant extract is Cistanche deserticola total glycoside extract.

[0025] The present invention has the following beneficial effects:

[0026] (1) Compared with the traditional affinity ultrafiltration-mass spectrometry technology for screening active ingredients, the present invention optimizes the subject of affinity ultrafiltration screening, uses drug-containing brain tissue matrix instead of Chinese medicine extracts, and the formation of protein-ligand complexes occurs under conditions that simulate the degrees of freedom in actual biological systems, making this screening method more practical and reliable.

[0027] (2) Potential MAO-B inhibitors derived from plants have the characteristic of natural low toxicity. The optimized affinity ultrafiltration-liquid chromatography-mass spectrometry screening system of the present invention is of great significance for rapidly discovering target compounds that can cross the blood-brain barrier from natural medicines.

[0028] (3) As a high-throughput method, the method of the present invention is rapid, simple, stable, highly precise and reproducible. The screening method of the present invention can more effectively reflect the potential ligand information with MAO-B inhibitory effect in plants, and the selected ultrafiltration membrane pore size provides a better separation effect.

[0029] (4) Compared with traditional screening procedures, affinity ultrafiltration coupled with chromatography is not only very simple but also very effective, greatly reducing the time, sample, and reagent usage required for screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the total ion current diagram of the dissociation solution of the blank control group;

[0031] Figure 2 This is the total ion current diagram of the dissociation solution of the experimental group;

[0032] Figure 3 This is the total ion current graph of the dissociation solution of the inactivated protein control group;

[0033] Figure 4 is the peak area of ​​each component in affinity ultrafiltration;

[0034] Figure 5 is the IC50 of each potential inhibitor. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples and drawings. It should be understood that these examples are only for illustrative purposes and in no way limit the scope of protection of the present invention.

[0036] Example 1

[0037] Weigh 5 kg of crushed Cistanche deserticola medicinal material, heat and reflux extract with 70% ethanol aqueous solution (10 times the amount) for 3 times, each time for 2 hours, combine the extracts after extraction, concentrate under reduced pressure at 60°C to form a thick paste, purify using a macroporous resin (D101) chromatographic column, and elute with water and 50% ethanol aqueous solution by volume, collect the fractions eluted with 50% ethanol by volume, concentrate, and dry under a vacuum environment at 60°C to obtain the total glycoside extract of Cistanche deserticola (stored in a refrigerator at -20°C for future use).

[0038] Through in vitro MAO-B inhibitory activity detection, the results were calculated according to formula (1), and the MAO-B inhibition rate of the total glycosides extract of Cistanche deserticola was 1.543±0.106 mg / mL, confirming that the extract contained MAO-B inhibitory components, and the next experiment was carried out.

[0039] MAO-B inhibition rate = [1-(A Drugcontrol -A Drug ) / (A Control -A Standard )]×100% (1)

[0040] Among them, A Control A is the absorbance value of the non-reactive well with PBS+kynurenine. Standard is the absorbance value of the reaction well of kynurenamine and MAO-B, A Drugcontrol is the absorbance value of the unreacted drug well of drug+kynurenine, A Drug is the absorbance value of the reaction well containing drug + MAO-B + kynurenamine.

[0041] (2) Male SD rats were given plant extracts at a dosage of 10 mL / kg / d. Three days after intragastric administration, the drug-containing rat brain tissue was obtained;

[0042] (3) The drug-containing rat brain tissue was homogenized, then centrifuged at 3000 rpm for 10 min, and the supernatant was vortexed with three times the amount of acetonitrile for 5 min to precipitate protein; then centrifuged at 12000 rpm for 10 min, and the supernatant was dried. Finally, the residue was dissolved in 400 μL PBS to obtain the drug-containing brain homogenate extract.

[0043] Example 2

[0044] An experimental group, an inactivated protein control group, and a blank control group were set up. MAO-B solution was added to the experimental group, the inactivated protein control group replaced the MAO-B solution with the inactivated MAO-B solution, and the blank control group replaced the MAO-B solution with the PBS solution to exclude interference from the control, false positives, and nonspecific binding. The specific steps are as follows:

[0045] (1) 100 μL of drug-containing brain homogenate extract and 20 μL of MAO-B (the control group used 20 μL of inactivated MAO-B, and the blank group used 20 μL of PBS) were mixed evenly and incubated at 37°C for 60 min to allow MAO-B to bind to the active ingredients in the drug-containing brain homogenate extract.

[0046] (2) The incubated mixture was transferred to a 30 kDa ultrafiltration centrifuge tube, and centrifuged at 12,000 rpm for 15 min in a centrifuge at 4°C. The filtrate was discarded. Then, 200 μL of PBS solution was added to the upper layer of the ultrafiltration centrifuge tube, and the mixture was mixed and centrifuged at 12,000 rpm for 15 min in a centrifuge at 4°C. The filtrate was discarded. The operation was repeated three times to obtain a conjugate.

[0047] (3) Add 200 μL of 70% methanol aqueous solution (dissociation agent) to the obtained complex, mix well and let stand for 10 min, then centrifuge at 12,000 rpm for 15 min at 4°C, collect the filtrate, and repeat the operation 3 times; dry the filtrate with a nitrogen blower to obtain a dissociation liquid residue.

[0048] (4) The residue of the dissociation solution was redissolved in 400 μL of 50% methanol aqueous solution, filtered through a 0.22 μm microporous membrane, and centrifuged at 12,000 rpm for 30 min. The supernatant was then used for UPLC-Q-Exactive-MS analysis.

[0049] Liquid phase conditions: Chromatographic column: ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm; Waters, USA); column temperature: 30°C; flow rate: 0.2 mL min-1; injection volume: 3 μL; mobile phase: phase A is acetonitrile; phase B is water containing 0.1% formic acid;

[0050] Gradient elution program: 0-8 min, 2%-12% A; 8-16 min, 12-16% A; 16-19 min, 16% A; 19-22 min, 16%-19% A; 22-25 min, 19% A; 25-30 min, 19%-25% A; 30-32 min, 25% A; 32-35 min, 25%-30% A; 35-38 min, 30%-40% A; 38-40 min, 40%-50% A; 40-45 min, 50%-90% A; 45.2-50 min, 2% A.

[0051] The mass spectrometry conditions were as follows: electrospray ionization source (ESI); negative ion mode; capillary voltage 3500 V; capillary temperature 350°C; sheath gas N2: 45 unit, auxiliary gas N2: 15 unit; collision energy: 10, 30, 50 eV; detection method: Full MS / ddMS2 (Full MS resolution is 70,000 FWHM, dd-MS2 resolution is 17,500 FWHM); mass scanning range: m / z 100-1500; S-Lens RF Level: 55.

[0052] Example 3

[0053] (1) MAO-B inhibition rate verification

[0054] The potential ligands are separated and identified by phytochemical methods and modern spectral techniques (or directly purchased from commercial sources). The MAO-B inhibition rate is determined by measuring the absorbance change trend of 4-hydroxyquinoline, the product of kynurenine catalyzed by MAO-B, under 315nm excitation and 380nm emission. The components of the inhibition rate determination reaction system are: MAO-B (20μL, 0.5mg / mL blank group does not contain monomers), kynurenine (50μL, 1mM), and the sample to be tested (130μL, 0.1μΜ-1mM). The total reaction volume is 200μL. After all components were mixed evenly in advance, they were incubated at 37°C for 30 minutes, and then 10% TCA was added in a 1:1 ratio to terminate the reaction. After standing for 10 minutes, 2M NaOH was added for color development, and the OD value at 315nm excitation and 380nm emission was immediately recorded using an ELISA reader. Rasagiline (a marketed MAO-B inhibitor drug) was used as a positive control. Each sample was measured in parallel 3 times. The MAO-B inhibition rate was calculated using formula (1).

[0055] (2) Experimental results

[0056] Affinity ultrafiltration-liquid chromatography-mass spectrometry technology screened out 5 potential ligands from the drug-containing brain homogenate extract, all of which had MAO-B inhibitory activity.

[0057] The active enzyme has different affinities for the drug-containing brain homogenate extract compared to the inactivated enzyme. Based on the difference in affinity between the ligand-enzyme complex after incubation, the potential ligands in the active enzyme group showed larger peak areas than those in the inactivated enzyme group. In order to further evaluate the binding strength between the ligand and the receptor, the relative binding affinity and specific binding rate were used to compare the changes in the relevant peak areas in the affinity ultrafiltration total ion current graph before and after inactivation. The calculation formula is:

[0058] Specific binding value (SBV) = relative peak area of ​​experimental group / relative peak area of ​​denaturation group

[0059] Binding rate (BR) (%) = (peak area of ​​experimental group - peak area of ​​inactivated protein control group) / peak area of ​​blank group × 100%.

[0060] The relative peak area of ​​the experimental group and the relative peak area of ​​the inactivated protein group represent the TIC peak areas of active and inactivated MAO-B obtained from the drug-containing brain homogenate, respectively. Table 1 lists the potential binding ligands. According to the results of liquid chromatography-mass spectrometry identification, Table 1 shows.

[0061] The potential ligands screened out in the affinity ultrafiltration were preferentially subjected to MAO-B inhibition rate determination according to formula (1). The inhibition rates of the potential ligands are shown in Table 1, which are: echinacoside 179±36.351 μM, verbascoside: 119.9±5.881 μM, isoverbascoside: 86.037±7.343 μM, tubuloside B: 132.7±16.208 μM, and 2'-acetylverbascoside 17.71±5.599 μM.

[0062] Table 1

[0063]

Claims

1. A method for high-throughput screening of monoamine oxidase B inhibitors, characterized in that: The following steps are involved: (1) homogenizing drug-containing rat brain tissue, centrifuging once to precipitate protein, centrifuging twice, taking the supernatant and blowing it dry with nitrogen, and then dissolving the obtained residue in sodium phosphate buffer solution (PBS) to obtain a drug-containing brain homogenate extract; (2) incubating the drug-containing brain homogenate extract with monoamine oxidase B, and then ultrafiltering the mixture to obtain a conjugate; (3) adding a 70% by volume methanol aqueous solution to the combined body to dissociate, collecting the dissociation liquid by centrifugation and drying to obtain a dissociation liquid residue; (4) The residue of the dissociation solution was dissolved in a 50% by volume methanol aqueous solution, filtered through a microporous membrane, and analyzed and identified by UPLC-Q-Exactive-MS to obtain a monoamine oxidase B inhibitor.

2. A method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: The volume ratio of drug-containing brain homogenate extract to monoamine oxidase B is 5:

1.

3. The method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: The incubation time in step (2) is 1 h and the incubation temperature is 37°C.

4. The method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: In step (3), the dissociation time is 5 min and the incubation temperature is 37°C.

5. The method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: The liquid phase conditions for analysis and identification in step (4) are: Chromatographic column: ACQUITY HSS T3 (2.1×100 mm, 1.8 μm; Waters, USA); column temperature: 30°C; flow rate: 0.2 mL·min -1 ; Injection volume: 3 μL; Mobile phase: Phase A is acetonitrile; Phase B is water containing 0.1% formic acid; Gradient elution.

6. The method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: The gradient elution program was as follows: 0-8 min, 2%-12% A; 8-16 min, 12-16% A; 16-19 min, 16% A; 19-22 min, 16%-19% A; 22-25 min, 19% A; 25-30 min, 19%-25% A; 30-32 min, 25% A; 32-35 min, 25%-30% A; 35-38 min, 30%-40% A; 38-40 min, 40%-50% A; 40-45 min, 50%-90% A; 45.2-50 min, 2% A.

7. The method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: The mass spectrometry conditions for analysis and identification in step (4) are as follows: electrospray ionization source (ESI); negative ion mode; capillary voltage 3500 V; capillary temperature 350°C; sheath gas N2: 45 unit, auxiliary gas N2: 15 unit; collision energy: 10, 30, 50 eV; detection method: Full MS / ddMS2 (Full MS resolution is 70,000 FWHM, dd-MS2 resolution is 17,500 FWHM); mass scanning range: m / z 100 to 1500; S–Lens RF Level:

55.

8. The method for high-throughput screening of monoamine oxidase B inhibitors according to claim 1, characterized in that: The potential inhibitors of monoamine oxidase B are echinacoside, verbascoside, isovalascoside, tubuloside B and 2'-acetylverbascoside.

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