CN116768835A - Monoamine oxidase B inhibitor and application thereof in medicines for resisting neurodegenerative diseases - Google Patents

Abstract

The application discloses a monoamine oxidase B inhibitor and application thereof in medicines for resisting neurodegenerative diseases. The monoamine oxidase B inhibitors comprise sedanolactone and neocnidilide. The sirtuin lactone and the neocnidilide can reversibly inhibit the activity of human monoamine oxidase B and IC ₅₀ 103nmol/L and 405nmol/L respectively, and by measuring the activity of human neuroblastoma cells, the neuroprotection of the sedanolactone was comparable to that of the positive control rasagiline. The single phthalide compound sedanolide and neocnidilide provided by the application can be used as safe and effective natural product components, provides a theoretical basis for preparing medicaments for treating neurodegenerative diseases, and is expected to be popularized and used as a new monoamine oxidase B inhibitor type anti-neurodegenerative medicament.

Description

A monoamine oxidase B inhibitor and its use in anti-neurodegenerative disease drugs application

Technical field

The present invention relates to the fields of natural product activity screening and neurodegenerative disease medicine, specifically a monoamine oxidase B inhibitor and its application in anti-neurodegenerative disease drugs.

Background technique

Parkinson’s Disease (PD), also known as paralysis shaking, is the second most common progressive neurodegenerative disease in the world and the most common extrapyramidal disease in middle-aged and elderly people. In my country, the prevalence of PD in people over 65 years old is approximately 1.7%, with a total population of 3 million, accounting for approximately 50% of the total number of patients worldwide. The pathological basis is mainly the degeneration and death of dopaminergic neurons in the substantia nigra pars compacta of the midbrain.

Monoamine Oxidase B (MAO-B) is a flavin-containing adenine dinucleotide that is widely distributed on the outer membrane of mitochondria in central and peripheral tissues and regulates the metabolism and distribution of monoamine neurotransmitters. (FAD) enzyme. MAO-B is a key enzyme that degrades dopamine in the body. The cysteine residue in the active center of its coenzyme FAD can react with dopamine to eventually produce ammonia, aldehydes and hydrogen peroxide. Overexpression or increased activity of MAO-B is an important factor in the pathogenesis of PD. Increased MAO-B activity in brain tissue is one of the indicators of brain function decline. The neuroprotective effect of MAO-B inhibitors is particularly prominent. By reducing the generation of free radicals in the brain, it exerts a protective effect on the central nervous system and can improve patients' motor symptoms. Long-term use will not cause mental disorders, movement complications, or dyskinesias. and other adverse reactions, and is especially suitable for the treatment of degenerative diseases of the central nervous system such as PD and Alzheimer's disease. However, commercial MAO-B inhibitors are mainly selegiline, rasagiline and safinamide, with less clinical selectivity. Rasagiline is the most commonly used in my country, but because it is an irreversible inhibitor, there are Certain side effects and drug resistance.

Natural product extracts are a rich source for drug development against this neurodegenerative disease. The discovery of bioactive ingredients with MAO-B inhibitory effects from traditional Chinese medicine is crucial in the development of drugs for neurodegenerative diseases. Currently, the main methods for screening MAO-B inhibitors from natural products include activity tracking separation methods, magnetic bead ligand fishing methods, affinity ultrafiltration methods, and online post-column bioactivity assays. However, they all have low throughput and compound limitations. Due to the technical defect of asynchronous separation and activity evaluation, the present invention is based on the MAO-B inhibitory micro-fraction activity screening technology and discovered monophthalide compounds-cerdanolactone and neostantiolide from the traditional Chinese medicine Ligusticum chuanxiong. According to Relevant literature reports that it has rich biological activities such as anti-inflammatory, sedative, and anti-cardiovascular disease, but its activity in inhibiting MAO-B has not yet been reported.

Contents of the invention

In order to overcome the shortcomings and shortcomings of the prior art, the primary purpose of the present invention is to provide a monoamine oxidase B inhibitor. The active ingredients of the monoamine oxidase B inhibitor are cerdanolactone and neostantiolide, which are extracted from the natural product Ligusticum chuanxiong extract. Through biological activity testing, it was found that cerdanolide and neoostidolide have excellent MAO-B inhibitory activity. They are a type of highly active MAO-B inhibitors that can inhibit recombinant human monoamine oxidase B. SH-SY5Y nerve cell activity tests have shown good neuroprotective effects, and it is expected to be promoted and used as a new monoamine oxidase B inhibitor anti-neurodegenerative disease treatment drug.

The second object of the present invention is to provide a monoamine oxidase B inhibitor and its application in anti-neurodegenerative disease drugs.

The primary purpose of the present invention can be achieved through the following technical solutions:

A monoamine oxidase B inhibitor, containing a monophthalide compound of formula I and/or formula II,

Preferably, the formula I is Sedanolide (also known as 3-butyl-3a,4,5,6-tetrahydro-1(3H)-isobenzofuranone, CAS: 6415-59 -4); the formula II is Neocnidilide, also known as (3S,3aR)-3-butyl-3a,4,5,6-tetrahydro-1(3H)-isobenzofuran Ketone or (3S, 3aR)-(-)-cerdanolactone, CAS: 4567-33-3); the formula I and formula II are phthalide isomers.

Preferably, the monoamine oxidase B inhibitor is a human monoamine oxidase B inhibitor.

Preferably, the monophthalide compound of formula I and/or formula II is derived from Ligusticum chuanxiong extract.

Preferably, the monophthalide compounds of formula I and/or formula II are discovered and screened from Ligusticum chuanxiong through micro-fraction activity screening technology based on MAO-B inhibitory properties.

Preferably, the specific screening steps for the monophthalide compounds of Formula I and/or Formula II are as follows:

20 μL of Ligusticum chuanxiong semi-prepared segmented samples were separated and analyzed using plate-joining liquid phase conditions on a microfluidic activity screening platform, and collected in a 384 black microplate. Using the 384 microwell plate activity detection conditions, the Ligusticum chuanxiong semi-prepared sample was tested with a microplate reader to obtain Ligusticum chuanxiong semi-prepared samples. The monoamine oxidase B inhibitory activity spectrum of the sample was prepared, and then mass spectrometry liquid phase conditions and mass spectrometry conditions were used to perform LC-MS analysis on the semi-prepared sample of Ligusticum chuanxiong. Based on the activity spectrum and LC-MS data, Cerdan was screened out from Ligusticum chuanxiong. Acidolactone and/or neocniolactone.

Preferably, the connecting plate liquid phase conditions: Shimadzu LC-20AT high-performance liquid phase system, chromatographic column: medium spectrum RD-C18 (5 μm, 4.6 × 250 mm), flow rate: 0.5 mL/min; detection wavelength: 220 nm, 254 nm , 280nm; mobile phase: phase A - 0.1% formic acid - water, phase B - 0.1% formic acid - methanol; injection volume: 20 μL; column temperature: 30°C; elution conditions: 0-30min, 55%-70% B , 30-60min, 70%-80% B, 60-61min, 80-100% B.

Preferably, the 384 microwell plate activity detection conditions: add 25 μL of 16 μg/mL MAO-B enzyme solution to each well of the dry sample plate, centrifuge at 2000 rpm for 5 min, and add 25 μL of kynurenine dihydrogen with an initial concentration of 92 μM. Bromate (Kynuramine, KYN) substrate solution, centrifuge at 2000 rpm for 2 minutes, transfer to Biotek Synergy H2 microplate reader, incubate at 37°C for 10 minutes, set the fluorescence excitation wavelength to 310nm, and the emission wavelength to 370nm for 1 hour kinetic detection. The kinetic curve was obtained, the signal growth slope was calculated, and the biological activity spectrum was constructed based on the slope-liquid phase data using Origin 9.0 software. The above experiment was repeated twice.

Preferably, the mass spectrometry liquid phase conditions: Thermo Scientific SII high-performance liquid phase system, chromatographic column: mid-spectrum RD-C18 (5 μm, 4.6 × 250 mm); flow rate: 0.5 mL/min; PDA detector (190-800 nm), Detection wavelength: 280, 254, 220nm; Mobile phase: Phase A-0.1% formic acid-water, Phase B-0.1% formic acid-methanol, injection volume: 2μL; column temperature: 30°C; elution conditions: 0-30min, 55%-70% B, 30-60min, 70%-80% B, 60-61min, 80-100% B.

Preferably, the mass spectrometry conditions: Thermo Scientific Orbitrap Exploris 120 mass spectrometry system, application mode: small molecules, ion type: H-ESI; spray voltage: static; positive ions: 3500V; negative ions: 3000V; gas mode: static; sheath gas : 50Arb; auxiliary gas: 10Arb; scavenge gas: 1Arb; ion transfer tube temperature: 320°C, evaporator temperature 400°C, and leucine enkephalin was used as the internal standard to calibrate the mass accuracy.

Level 1 scan: full scan; Orbitrap resolution: 60000; Scan range (m/z): 100-1000; RF lens (%): 70; Scan both positive and negative ions; Level 2 scan: Isolation window (m/z ): 1.5; Collision Energy Type: Normalized; HCD Collision Energy (%): 20, 50, 80; Orbitrap Resolution: 15000; Scan Range Mode: Automatic.

The second object of the present invention can be achieved through the following technical solutions:

The use of a monoamine oxidase B inhibitor as an anti-neurodegenerative disease drug.

Preferably, the anti-neurodegenerative disease drugs are tablets, dropping pills, sublingual tablets, injections, granules, capsules, oral liquids, microcapsule microsphere preparations, sprays, targeted preparations, and powder injections. or at least one of aerosols.

Preferably, the anti-neurodegenerative diseases are central nervous system disorders, psychiatric disorders, neurodegenerative disorders and pain disorders, such as neurodegenerative diseases, cardiovascular diseases, stroke and stroke.

Preferably, the monoamine oxidase B inhibitor increases the content of dopamine in the body by inhibiting the activity of monoamine oxidase B, thereby achieving a therapeutic effect on neurodegenerative diseases.

Specifically, the monoamine oxidase B inhibitor exerts a neuroprotective effect by inhibiting monoamine oxidase B in human neuroma cell blasts to achieve an anti-neurodegenerative disease effect. The monophthalide compound of Formula II and the second generation monoamine oxidase B inhibitor drug The neuroprotective effects of rasagiline are consistent.

Preferably, the anti-neurodegenerative disease drug is a pharmaceutical composition for preventing and/or treating Parkinson's disease and its complications.

Furthermore, through Parkinson's cell activity detection, it was investigated that serdanolide and neoostidolide, as MAO-B inhibitors, can resist apoptosis caused by 6-OHDA neurotoxicity in nerve cells SH-SY5Y. , exerting a neuroprotective effect.

The monoamine oxidase inhibitors disclosed in the present invention include monophthalide compounds. The monophthalide compounds are cerdanolide and neostantiolide, which are a pair of isomers derived from the extract of the traditional Chinese medicine Ligusticum chuanxiong. body compounds. In previous studies, only the anti-inflammatory, sedative and other activities of cerdanolide and neoostricholide were discovered and applied, but there were no specific studies on the inhibitory activity of monoamine oxidase B, a neurodegenerative target, and this study After applying for a large number of experiments, it was found that the compounds cerdanolide and neoostylide have obvious inhibitory effects on human monoamine oxidase B. Experiments have found that the inhibitory activities of the compounds cerdanolide and neoostidolide on monoamine oxidase B are 103 nmol/L and 405 nmol/L respectively, and they show reversible inhibition of human monoamine oxidase B. In addition, the compounds serdanolide and neostanniolide both showed dose-dependent neuroprotective effects on Parkinson's SH-SY5Y cells, and the neuroprotective effect of the compound serdanolide was comparable to that of rasagiline. Therefore, the compounds cerdanolide and neoostidolide provided by the present invention can be used as safe and effective natural product components, provide a theoretical basis for the preparation of drugs for the treatment of neurodegenerative diseases, and are expected to be used as a new monoamine oxidase B Promotion and use of inhibitor anti-neurodegenerative disease drugs.

Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

(1) The compound cerdanolide or neoostidolide according to the present invention has a novel structural type as a monoamine oxidase inhibitor;

(2) The compounds of the present invention, cerdanolide or neoostidolide, have good inhibitory activity and reversible inhibitory properties on monoamine oxidase. After acting on Parkinson's cell models, they show similar effects to the second-generation commercial drug rasa. Guillen has considerable neuroprotective effects;

(3) The present invention can further optimize the structure of the better compounds obtained through screening, thereby improving its inhibitory activity and selectivity against monoamine oxidase B;

(4) The monoamine oxidase B inhibitor prepared by the present invention has good application prospects in medicine for treating degenerative diseases of the central nervous system.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 is a diagram of the micro-fraction activity screening platform for monoamine oxidase B inhibitors in Example 1 of the present invention;

Figure 2 is a monoamine oxidase B inhibitor screening activity profile diagram of a semi-prepared sample of Ligusticum chuanxiong in Example 3 of the present invention;

Figure 3 is the mass spectrum of compounds I and II identified from semi-prepared samples of Ligusticum chuanxiong in Example 4 of the present invention;

Figure 4 is a comparison chart between the liquid phase peaks of compounds I and II of Example 5 of the present invention and the activity peak of Ligusticum chuanxiong semi-prepared sample;

Figure 5 is a fitting diagram of the half inhibitory concentration of monoamine oxidase B of compounds I, II and rasagiline in Example 6 of the present invention;

Figure 6 is a diagram showing the reversibility study results of compounds I and II inhibiting MAO-B in Example 7 of the present invention;

Figure 7 is a diagram showing the results of the neuroprotective effects of compounds I and II on SH-SY5Y cells in Example 8 of the present invention.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples are intended to be illustrative only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

For the first time, the present invention discovered two phthalides and their new applications from Ligusticum dechuanxiong based on the monoamine oxidase B microfraction inhibitory activity screening platform, that is, the application of two phthalides in the preparation of anti-neurodegenerative disease drugs.

Example 1

Monoamine oxidase B microfluidic inhibitory activity analysis, isolation and screening platform

Based on the pipeline modification of the Shimadzu LC-20AT high-performance liquid phase system, the entire platform mainly includes the separation analysis and detection part and the fraction collection part, as shown in Figure 1.

(1) Separate the analysis and detection part, use a 250 μm inner diameter Peek tube to connect the high-performance liquid phase quaternary pump, manual valve, and chromatographic column part, and then use tees and 120 μm inner diameter Peek tubes of different lengths to connect the components flowing behind the column. Split the flow and change the pressure in the pipeline by adjusting the length of the two Peek tubes at the tee outlet, thereby changing the flow rate in the pipeline so that about 1/3 of the post-column flow flows to detectors such as DAD and MS. , about 2/3 flows to the fraction collection device.

(2) In the fraction collection part, the pipeline is fixed on the mechanical arm of a CTC HTS PAL (liquid multifunctional fully automatic sampler system) for automatic sampling, and the fraction collection point at the end of the sampling outlet is The device is composed of a two-way, connector and other accessories connected to a capillary tube with an inner diameter of 100 μm and an approximate length of 8 cm. Using the debugged software PAL Cycle composer and Drivers of the CTC HTS PAL automatic sampling system, by executing the written programming language, the robotic arm and the spotter fixed on it can accurately move in parallel on the x, y, and z axes. , the translation distance accuracy can reach up to 0.1mm, and the residence time frequency of a single hole in the orifice plate can reach up to 0.1s/hole.

(3)Workflow of biological activity testing

The sample enters the micro-fraction activity analysis and screening platform. It first enters the detection module DAD to detect the peak state of the substance. The corresponding fractions are collected into the micro-well plate through fractionation. Due to the presence of organic solvents in the mobile phase of the well plate, it is bioincompatible with the enzyme. Gradient elution of complex and complex systems brings problems such as non-uniformity of organic solvent content. Therefore, the microplate with collected fractions needs to be placed in a vacuum drying oven to dry to remove water, organic solvents, and formic acid in the mobile phase. Wait for interfering solvents without destroying the chemical structure of the compounds remaining in the microwell plate; after removing the interfering solvents, take out the dry black microwell plate and use a multi-channel electronic pipette to add enzyme and substrate solutions to the well plate in sequence; The loaded microplate is then placed in a multifunctional microplate reader for kinetic testing to achieve activity assessment.

(4) Principle of biological activity detection

Based on the oxidative deamination characteristics of monoamine oxidase, and taking into account the need for high-throughput activity detection in complex systems - that is, simple operation, high sensitivity, high signal-to-noise ratio, and adaptability to micro-fraction activity analysis and screening platforms, etc., In the present invention, kynurenamine dihydrobromide (KYN) is selected as the substrate of MAO-B. MAO-B can oxidatively deaminate the methyl group of KYN to form the fluorescent product 4-Hydroxy Quinoline. 4-HQ), the product 4-HQ produces highly sensitive fluorescence signals at an excitation wavelength of 310nm and an emission wavelength of 370nm, which is used to test the activity of MAO-B.

This system is based on the fact that the same amount of substrate is oxidized and deaminated by MAO-B at the same time to form a product. The activity of MAO-B is expressed by the rate of kinetic detection of the fluorescence signal of the product. The absence of MAO-B inhibitor is used as a control. And/or the linear growth rate (slope) of the fluorescence signal of the product generation rate without inhibitors can be used to determine whether there are inhibitory components in the system and the strength of the inhibitory effect.

(5) Construction of activity map

Sort each well on the microplate according to the sequence of fraction collection, and calculate the fraction collection time corresponding to each well based on the log. In the microplate in which the sample is collected, the slope values of the fluorescence growth kinetic curve for each well on the plate are arranged in the numbered order mentioned above. Taking the time point when the well plate is detected in the microplate reader as the x-axis, and taking the signal obtained by a single well at different times as the y-axis, use the slope formula of the Excel table to calculate the growth slope of the signal detected in each well of the microplate. The slope of each well of the microplate is arranged in order of plate connection time. Then find the median of all slope values, and then divide all slope values by the obtained median to normalize all growth slopes to facilitate parallel comparisons between multiple experimental test results. Finally, with the fraction collection time as the x-axis and the corresponding standardized slope value as the y-axis, Origin 9.0 software was used to combine the reconstructed biological activity spectrum with the DAD chromatogram and mass spectrum total ion chromatogram obtained from each parallel detection through fractionation. By correlating and integrating the collection time, biological information and chemical information can be obtained simultaneously, and the chemical structure information of the biologically active components in the picture can be analyzed.

Example 2

Isolation, extraction and semi-preparation of Ligusticum chuanxiong

(1) Crushing, extraction and separation of Ligusticum chuanxiong: Ligusticum chuanxiong is a crude extract extracted by ultrasonic extraction with 70% ethanol. The specific extraction process is: take a certain amount of the traditional Chinese medicine Ligusticum chuanxiong and place it in a clean oven, bake it at 35°C for 24 hours to remove moisture, then crush the medicinal material, pass it through a 24-mesh sieve, collect the sieved material, and weigh 12.0g of the processed medicinal material powder or granules. , placed in a 500mL Erlenmeyer flask, material-to-liquid ratio (mL/g): 1:25, add 300mL of 70% ethanol, shake well, soak overnight, extraction temperature: 30°C, ultrasonic for 60 minutes, power 360W, extraction 3 After filtration, the extracts were combined, the solvent was evaporated by rotary evaporation at 45°C, and concentrated to obtain a crude extract. Store in a refrigerator at -20°C away from light.

(2) Preparation of liquid phase samples: Dissolve the crude extract of Ligusticum chuanxiong into a high-concentration mother solution, dilute it with 70% methanol to prepare a sample solution of 50 mg/mL, and filter it with a 0.22 μm organic micropore filter. Perform membrane filtration and collect the filtrate as a semi-prepared sample solution. Semi-preparative liquid chromatography was used to sample 50 mg/mL Ligusticum chuanxiong 70% methanol sample according to the peak appearance.

(3) Semi-preparative liquid phase conditions: Agilent 1200series liquid phase system, medium spectrum RD-C18 (5μm, 10.0×250mm); flow rate: 3mL/min, detection wavelength: 254nm, mobile phase: Phase A-0.1% formic acid-water , Phase B—0.1% formic acid-methanol, injection volume: 500 μL. Elution conditions: 0-30min, 40%-80%B, 30-45min, 80%-90%B, 45-55min, 90%-100%B, 55-75min, 100%B.

(4) Collection and concentration of semi-preparation segmented liquid: According to the preliminary test results, collect the post-column effluent in the 16-25 min period, and rotary evaporate the liquid phase segmented liquid at 45°C to obtain a crude extract, methanol/ Re-dissolve in water, transfer to a brown glass sample bottle, evaporate the solvent in a 35°C vacuum drying oven, seal and store in a -20°C refrigerator.

Example 3

Dose-effect relationship spectrum of monoamine oxidase B activity inhibition of semi-prepared samples of Ligusticum chuanxiong

Protein source: Human monoamine oxidase B (MAO-B) pure protein (liquid, 0.5 mL, concentration 5 mg/mL), recombinant protein expressed in baculovirus-infected BTI insect cells, product number M7441, sourced from Sigma Company, preserved Store in -80°C refrigerator.

(1) Preparation of samples

Semi-prepared solution of Ligusticum chuanxiong: Take the segmented crude extract of semi-prepared liquid phase of Ligusticum chuanxiong, add 1mL of 70% methanol to prepare a higher concentration injection solution, pass it through a 0.22μm organic microporous filter membrane, and take the continued filtrate as the liquid phase for high concentration injection. sample solution; then dilute it with 70% methanol to half of the original concentration to serve as a liquid phase injection solution.

Preparation of PBS buffer: Weigh a certain amount of disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride and sodium chloride, add ultrapure water, and prepare a PBS buffer with a concentration of 100mmol/L, and adjust the pH to 7.4 .

Enzyme solution: Take out 20 μL of the aliquoted monoamine oxidase B stock solution from the -80°C refrigerator, transfer it to a 37°C water bath and incubate it for 10 minutes, and dilute it with PBS buffer to make an enzyme solution of 16 μg/mL.

Substrate solution: Precisely weigh a certain amount of substrate KYN, add an appropriate amount of PBS buffer to dissolve the substrate, sonicate for 15 minutes, prepare a high-concentration mother solution, and dilute it with PBS buffer to form a substrate solution of 92 μmol/L.

(2) Chromatographic conditions: Shimadzu LC-20AT high-performance liquid phase system, chromatographic column: mid-spectrum RD-C18 (5μm, 4.6×250mm), flow rate: 0.5mL/min; detection wavelength: 220nm, 254nm, 280nm,; flow Phase: Phase A - 0.1% formic acid - water, Phase B - 0.1% formic acid - methanol; injection volume: 20 μL; column temperature: 30°C. Elution conditions: 0-30min, 55%-70% B, 30-60min, 70%-80% B, 60-61min, 80-100% B.

(3) Post-column microfluidic fraction collection: Inject 20 μL of sample into the microfluidic fraction screening system. After the sample is separated and analyzed by the liquid phase system, the post-column fraction is collected by a CTC automatic device at a frequency of 8s/well starting from the injection. In addition, in Before sample collection, add 10 μL of 10% DMSO aqueous solution to the 384 microwell plate. After the collection is completed, record the time of each well according to the single-well collection log; after collecting the sample, place the 384 microwell plate in a vacuum drying oven and vacuum at 35°C. Dry for 24h and remove the mobile phase;

(4) 384 microwell plate activity detection: Add 25 μL of 16 μg/mL MAO-B enzyme solution to each well of the dry sample plate, centrifuge at 2000 rpm for 5 min, add 25 μL of KYN substrate solution with an initial concentration of 92 μmol/L, and run at 2000 rpm. Centrifuge for 2 minutes, transfer to Biotek Synergy H2 microplate reader, incubate at 37°C for 10 minutes, set the fluorescence excitation wavelength to 310nm and the emission wavelength to 370nm for 1 hour of kinetic detection, obtain the kinetic curve, calculate the signal growth slope, and use Origin 9.0 The software constructed a biological activity spectrum based on the slope-liquid phase data, and the above experiment was repeated twice.

As shown in Figure 2, the activity peak of monoamine oxidase B is positively correlated with the injection concentration of the semi-prepared sample of Ligusticum chuanxiong. The liquid phase elution peak at 42 minutes corresponds to a larger activity peak of monoamine oxidase B, that is, the compound corresponding to this peak has a higher Monoamine oxidase B inhibitory activity.

Example 4

Mass spectrometry identification of natural monoamine oxidase B inhibitors in semi-preparative fractions of Ligusticum chuanxiong

The filtrate of the liquid phase semi-preparation segmented sample of Ligusticum chuanxiong that has passed through a 0.22 μm organic microporous filter membrane was injected into an ultra-high performance liquid chromatography-electrostatic orbital ion trap mass spectrometer for chromatographic analysis and structural identification.

(1) Sample preparation: Take 100 μL of Chuanxiong semi-prepared liquid-phase high-concentration injection solution to construct the activity spectrum, place it in a brown injection vial with an inner insert tube, and add 100 μL of 70 filtered by a 0.22 μm organic microporous membrane. The sample was diluted with % methanol (MeOH) as the mass spectrometry injection solution.

The setting conditions for each parameter in ultra-high performance liquid chromatography-electrostatic orbital ion trap mass spectrometry technology are as follows:

(2) Mass spectrometry conditions: Thermo Scientific Orbitrap Exploris 120 mass spectrometry system, application mode: small molecules, ion type: H-ESI; spray voltage: static; positive ions: 3500V; negative ions: 3000V; gas mode: static; sheath gas: 50Arb ; Auxiliary gas: 10Arb; Scavenge gas: 1Arb; Ion transfer tube temperature: 320°C, evaporator temperature 400°C, and leucine enkephalin was used as the internal standard to calibrate the mass accuracy.

Level 1 scan: full scan; Orbitrap resolution: 60000; scanning range (m/z): 100-1000; RF lens (%): 70; both positive and negative ions are scanned. Secondary scan: isolation window (m/z): 1.5; collision energy type: normalized; HCD collision energy (%): 20, 50, 80; Orbitrap resolution: 15000; scan range mode: automatic.

(3) Liquid phase conditions: Thermo Scientific SII high-performance liquid phase system, chromatographic column: mid-spectrum RD-C18 (5 μm, 4.6 × 250 mm); flow rate: 0.5 mL/min; PDA detector (190-800 nm), detection wavelength: 280, 254, 220nm; mobile phase: phase A-0.1% formic acid-water, phase B-0.1% formic acid-methanol, injection volume: 2 μL; column temperature: 30°C. Elution conditions: 0-30min, 55%-70% B, 30-60min, 70%-80% B, 60-61min, 80-100% B.

(4) Mass spectrometry identification of compounds I and II

Based on the chromatogram, primary mass spectrum, and secondary mass spectrum obtained by the electrostatic orbital ion trap high-resolution mass spectrometry, the ion chromatogram data was extracted for qualitative analysis of compounds I and II. Figure 3 is the 254nm chromatogram, total ion current mass spectrum and extracted ion chromatogram of the semi-prepared sample solution of Ligusticum chuanxiong in the positive ion mode in Example 4 of the present invention, and the fragmentation fragment diagram of compounds I and II; wherein, Figure A is Ligusticum chuanxiong The chromatogram of the semi-prepared sample solution at a wavelength of 254nm. Figure B is the total ion chromatogram of the semi-prepared sample solution of Ligusticum chuanxiong in the positive ion mode. Figure C is the extracted ion chromatogram of the molecular ion peak of 195 in the positive ion mode. Figure D These are the secondary mass spectrometry fragmentation fragment diagrams of compounds I and II obtained by screening in Example 1 of the present invention. As shown in A, B, and C in Figure 3, the retention times of compounds I and II are both 37.12 min, and their quasi-molecular ion peaks are m/z=195.1380[M+H]+. The inferred molecular formula is C ₁₂ H ₁₈ O ₂ . This ion further gave fragment ions 177.1273[M+HH ₂ O]+, 149.1322[M+HH ₂ O-CO]+, 125.0597[M+HC ₅ H ₁₀ ]+, 121.1010[M+HH ₂ O-CO-C ₂ H ₄ ]+, 107.0855 [M+HH ₂ O-CO-C ₃ H ₆ ]+, 93.0698 [M+HH ₂ O-CO-C ₄ H ₈ ]+. Therefore, the compounds I and II were identified as co-eluting phthalide isomers as cerdanolactone and neoostidolide.

Example 5

Positioning of liquid phase peaks and activity peaks of compounds I and II

Weigh a certain amount of standard compounds I and II, add DMSO to dissolve into a high-concentration mother solution, sonicate for 10 minutes, use 70% methanol to prepare a higher-concentration solution, filter with a 0.22 μm organic microporous membrane, collect the remaining filtrate, and take The filtrate was diluted with 70% methanol to prepare a 200 μmol/L liquid phase injection solution. The liquid phase conditions are the same as in Example 3.

As shown in Figure 4, the chromatographic peaks of compounds I and II are consistent with the 42-min chromatographic peak in the semi-preparation section of Chuanxiong and the elution time of the chromatographic peak analyzed by mass spectrometry. They all correspond to the same active inverted peak, that is, mass spectrometry analysis and chromatographic peak positioning. Compounds Ⅰ and Ⅱ under the same activity peak were analyzed to be cerdanolactone and neostantiolactone respectively.

Example 6

Inhibitory activity of compounds Ⅰ and Ⅱ on monoamine oxidase B

Use DMSO to dissolve compounds I and II to prepare high-concentration mother solutions, and dilute them with KYN substrate solution with an initial concentration of 92 μmol/L to prepare final concentrations of 78.125, 39.063, 19.531, 9.766, 1.953, 0.391, 0.195, 0.049, 0.024, 0.005, 0.001, 1.953E-04, 9.766E-05μmol/L substrate-containing inhibitor solutions (control DMSO content below 1%), and the substrate solution was used as a control, and the commercial drug rasagiline was used as a positive control.

MAO-B activity assay was performed in 384 black microplates. To evaluate the inhibitory activity of the sample to be tested, proceed based on the solution prepared above. Add 25 μL of the above solution to the 384 black microplate respectively, and add 25 μL of the 16 μg/mL enzyme solution with a volley gun, centrifuge at 2000 rpm for two minutes, and then set the Biotek Synergy H2 microplate reader at 37°C and the excitation wavelength of 310 nm. Kinetic detection was performed under the condition of emission wavelength 370nm. Each group of tests was measured three times in parallel to obtain the kinetic curve, calculate the signal growth slope, and calculate the inhibition rate. The change in fluorescence intensity is the reaction rate, and the inhibition rate calculation formula is:

Origin 9.0 software was used to plot and analyze the inhibition. The results are shown in Figure 5. The IC ₅₀ of compounds I and II for human monoamine oxidase B were 103 nmol/L and 405 nmol/L respectively. The commercial inhibitor rasagiline is 131 nmol/L. Compound Ⅰ has better inhibitory effect on human MAO-B than rasagiline.

Example 7

Kinetic experiments on reversible inhibition of monoamine oxidase B by compounds I and II

According to the IC ₅₀ of compounds I and II in Example 6, a series of different gradients (final concentrations: 5/4, 1, 3/4, 1/2 and 1/4 times the IC ₅₀ ) of inhibitor-containing PBS buffers were prepared. , add it to the 384 black microplate in the form of 18 μL/well. PBS buffer without inhibitors is used as a control. Add 16 μL/well of MAO-B enzyme with final concentrations of 6, 8, 10, 12, and 14 μg/mL. Solution, centrifuge for 2 minutes to mix, incubate at 37°C for 10 minutes, add 16 μL/well of substrate with a final concentration of 46 μM, centrifuge for 2 minutes to mix, place in Biotek microplate reader, 37°C, excitation wavelength and emission wavelength are 315 nm and 315 nm, respectively. Kinetic detection at 370nm for 1 hour. Plot reaction rate versus enzyme concentration. Measured in parallel three times.

The catalytic rates of compounds I and II at different concentrations were measured in MAO-B enzyme solutions of different concentrations. The ordinate of the compounds was fitted to the reaction rate, and the abscissa was the MAO-B enzyme concentration curve. As shown in Figure 6, the fitting curves of the reaction rates of MAO-B at different concentrations of compounds I (Figure 6-A) and II (Figure 6-B) all intersect at the origin. This model shows that compounds I and II Ⅱ are all reversible inhibitors of monoamine oxidase B.

Example 8

In vitro study on the neuroprotective effects of compounds Ⅰ and Ⅱ on SH-SY5Y cells

The purpose of this experiment is to detect the in vitro protective activity of the compound of the invention on nerve cells and further prove the medical use of the compound of the invention. This experiment uses 6-hydroxydopamine (6-OHDA) to induce the classic Parkinson's cell model, and the cell line used is human neuroma cell blast (SH-SY5Y) to test the protective activity of the inventive compounds I and II.

SH-SY5Y cells were cultured in DMEM culture medium (containing serum, 100 U/mL penicillin and 100 U/mL streptomycin), and cells were evenly seeded into a 96-well plate at a density of 1×10 ⁴ cells/mL and 100 μL/well. Incubate in a 37°C, 5% _CO2 incubator for 16 hours. When the cell density reaches about 80%, set up the modeling group, the administration group (compound I, compound II and the positive drug rasagiline) and the matrix group. Only the culture medium is added to the matrix group, and compound I, compound I and the drug administration group are added respectively. For compound II and positive control, the concentration gradient of the final concentration is 100 μmol/L, 20 μmol/L, 4 μmol/L, 0.8 μmol/L, 0.16 μmol/L, each group of 4 wells, DMSO is controlled within 0.1%, and incubated After 4 hours, the modeling group and the drug administration group were replaced with culture medium containing the dopamine neurotoxic drug 6-OHDA with a final concentration of 200 μmol/L, and the effect was continued for 24 hours. Use a microplate reader to measure the OD value at 450 wavelength, and use the CCK-8 method to evaluate the protective activity.

The results are shown in Figure 7. It can be clearly seen from Figure 7 that compounds I and II have significant dose-dependent protective activity on SH-SY5Y cells in the range of 0-20 μmol/L. Compound I and the positive drug rasagiline The protective effect is equivalent. Based on the above experiments, compounds I and II can exert a good neuroprotective effect in vitro and in vivo by reversibly inhibiting MAO-B, providing a reference for the treatment of Parkinson's disease.

Example 9

Tablet Preparation

Prescription (based on 1,000 tablets): 100g of pure compound formula I or II, 80g corn starch, 60g sucrose, 2g magnesium stearate. Preparation method: Mix the active ingredients with corn starch and sucrose, add water to moisten, stir evenly, dry, crush and sieve, add magnesium stearate, mix evenly, and press into tablets to obtain.

Example 10

Preparation of dropping pills

Prescription (based on the prescription amount of 1000 pills): 10g of pure compound formula I or II, 50g of polyethylene glycol (molecular weight 4000 or 6000), and appropriate amount of liquid paraffin. Preparation method: Heat polyethylene glycol-4000 or 6000 until it is completely melted, add the pure compound of formula I or II, stir and melt, keep it warm at 80-85℃, drop it from top to bottom until the liquid paraffin is cooled to 5-8℃ In, wait until the dropping pills are cooled and formed, take out the dropping pills, drain them and wipe off the liquid paraffin attached to the surface, put them in a bottle, and you have it.

Example 11

Preparation of sublingual tablets

Preparation of β-cyclodextrin inclusion complex: 10g of pure compound formula I or II, 80g of β-cyclodextrin, add β-cyclodextrin to 30°C water to make a saturated solution, and dilute the same amount of absolute ethanol The pure product of compound formula I or II is slowly dropped from top to bottom into the saturated solution of β-cyclodextrin, stirred at 30°C for 2 hours to include, shaped, taken out, cooled, refrigerated for 24 hours, washed and precipitated, dried at 40°C for 1 hour, and obtained.

Prescription (based on the prescription amount of 1,000 tablets): 60g of the above-mentioned β-cyclodextrin inclusion complex containing compound formula I or II, 165g of lactose, 6g of vitamin C, 15g of sodium carboxymethyl starch, 20g of povidone, and stearin 4g of acid, made into 1000 sublingual tablets according to conventional technology, obtained.

Example 12

Preparation of injection solutions

Prescription (based on 1,000 prescriptions): 100g of pure compound formula I or II, 100g of propylene glycol, and 1,000 mL of water for injection. Preparation method: Dissolve the pure compound of formula I or II in propylene glycol, add water for injection to 1000mL, mix evenly, filter, and divide the obtained solution into ampoules under sterile conditions to make 1mL/bottle for injection. Liquid, filled with nitrogen and sealed.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. A monoamine oxidase B inhibitor, characterized in that it contains a monophthalide compound of formula I and/or formula II, 2. The monoamine oxidase B inhibitor according to claim 1, characterized in that the formula I and formula II are phthalide isomers. 3. The monoamine oxidase B inhibitor according to claim 1, characterized in that the monoamine oxidase B inhibitor is a human monoamine oxidase B inhibitor. 4. The monoamine oxidase B inhibitor according to claim 1, characterized in that the monophthalide compound of formula I and/or formula II is obtained from Ligusticum chuanxiong through a micro-fraction activity screening technology based on MAO-B inhibitory properties. found and filtered out. 5. The monoamine oxidase B inhibitor according to claim 3, characterized in that the specific screening steps for the monophthalide compounds of formula I and/or formula II are as follows: 20 μL of Ligusticum chuanxiong semi-prepared segmented samples were separated and analyzed using plate-joining liquid phase conditions on a microfluidic activity screening platform, and collected in a 384 black microplate. Using the 384 microwell plate activity detection conditions, the Ligusticum chuanxiong semi-prepared sample was tested with a microplate reader to obtain Ligusticum chuanxiong semi-prepared samples. The monoamine oxidase B inhibitory activity spectrum of the sample was prepared, and then mass spectrometry liquid phase conditions and mass spectrometry conditions were used to perform liquid mass spectrometry analysis on the semi-prepared sample of Ligusticum chuanxiong. Based on the activity spectrum and liquid mass spectrometry data, formulas I and I were screened from Ligusticum chuanxiong. /or Formula II. 6. Application of the monoamine oxidase B inhibitor according to any one of claims 1 to 5 in anti-neurodegenerative disease drugs. 7. Application of the monoamine oxidase B inhibitor in anti-neurodegenerative disease drugs according to claim 6, characterized in that the anti-neurodegenerative disease drugs are tablets, dropping pills, sublingual tablets, and injections. , at least one of granules, capsules, oral liquids, microcapsule microsphere preparations, sprays, targeted preparations, powder injections or aerosols. 8. The application of the monoamine oxidase B inhibitor according to claim 6 in anti-neurodegenerative disease drugs, characterized in that the anti-neurodegenerative diseases are central nervous system disorders, psychiatric disorders, neurodegenerative disorders and Painful conditions such as neurodegenerative diseases, cardiovascular disease, stroke and stroke. 9. The application of the monoamine oxidase B inhibitor in anti-neurodegenerative disease drugs according to claim 6, characterized in that the monoamine oxidase B inhibitor increases the content of dopamine in the body by inhibiting the activity of monoamine oxidase B, thereby achieving anti-neurodegeneration. Therapeutic effect of diseases. 10. Application of the monoamine oxidase B inhibitor in anti-neurodegenerative disease drugs according to claim 6, characterized in that the anti-neurodegenerative disease drugs are used to prevent and/or treat Parkinson's disease and its complications. Pharmaceutical compositions.