Application of polyketone compound with special cage structure

Abstract

The invention relates to the technical field of microbial medicinal chemistry, in particular to medical application of a polyketone compound with a special cage structure, wherein the polyketone compound is separated from a fermentation product of a Penicillium daleae strain. The action potential discharge frequency and post-spontaneous excitatory post-prolapse current (sEPSCs) of hippocampus basteriform cell neurons are detected through a patch clamp technology, and it is proved that the compound has the remarkable activity of inhibiting neuronal excitatory and is non-toxic to nerve cells N2a under the active concentration. The compound can be used for preparing novel drugs for treating neuronal hyperexcitation related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pharmaceutical chemistry, and in particular to the use of a cage-structured polyketide compound derived from Penicillium daleae in the preparation of a drug for inhibiting excessive neuronal excitation. Background Art

[0002] Neurons are the basic structure and functional unit of the nervous system, which can receive, send and transmit electrical signals. Synapses are relatively special structures in neurons, which connect with surrounding nerve cells to form a neural network and are the structural basis for information transmission in organisms. Under normal circumstances, the presynaptic membrane releases neurotransmitters and binds to the receptors of the postsynaptic membrane to produce excitatory postsynaptic current (EPSC) or inhibitory postsynaptic current (IPSC), thus completing the signal conduction between the two neurons. When the excitatory electrical signals transmitted by the synapses of neurons continue to change, the cells will perform compensatory regulation to maintain the excitatory electrical signals transmitted by the synapses in a relatively stable state, which is called the homeostatic plasticity of the nervous system. When this homeostasis is destroyed and the neurons become overexcited, various diseases such as epilepsy and stroke will be caused.

[0003] Epilepsy is a chronic, recurrent syndrome of transient cerebral dysfunction, a neurological disease characterized by recurrent epileptic seizures caused by highly synchronized discharges of neurons. The main causes of epilepsy are trauma and recurrent epileptic seizures. Brain damage after an epileptic seizure can cause changes in the expression levels of various molecules that regulate the nervous system in brain cells, resulting in excessive neural proliferation and ectopic growth, causing another neuronal overexcitation leading to epileptic seizures.

[0004] At present, the main means of treating epilepsy is drug therapy, and inhibiting excessive neuronal excitation is the main target of drugs for treating epilepsy. Drugs used in the front line of clinical practice, such as carbamazepine, lamotrigine, gabapentin, etc., have many side effects and are prone to drug resistance. For example, carbamazepine can cause allergic rashes, while lamotrigine and gabapentin may cause dizziness, nausea, drowsiness, etc., and most anti-epileptic drugs have side effects on liver and kidney function and the blood system. Therefore, the development of new drugs that can inhibit excessive neuronal excitation and have fewer side effects has a very broad market prospect.

[0005] The applicant has extracted a variety of new compounds from the fermentation products of five fungi including Xylaria longifolia (see: Wang Qingyuan, Research on Chemical Composition and Biological Activity of Five Fungi Including Xylaria longifolia [D], South-Central University for Nationalities, Issue 03, 2024), including the cage-structured polyketide compounds described in the present invention. It is of great significance to study its pharmacological activity. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a cage-structured polyketide compound isolated from a fermentation product of Penicillium daleae for use in the preparation of a drug having the activity of inhibiting neuronal hyperexcitability.

[0007] The cage-like structure polyketone compound is a compound shown in Formula I:

[0008]

[0009] Furthermore, the drug having the activity of inhibiting neuronal overexcitability is a drug for the purpose of inhibiting neuronal overexcitability, such as a drug for treating epilepsy, stroke and Alzheimer's disease.

[0010] The cage-structured polyketide compound has the activity of inhibiting neuronal excitability, can be used as a lead compound for inhibiting neuronal overexcitation, and can be used for preparing drugs for inhibiting neuronal overexcitation.

[0011] The cage-structured polyketone compound of the present invention is soluble in dichloromethane, chloroform, acetone, methanol, pyridine, dimethyl sulfoxide and the like, but is insoluble in water.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] 1. The drugs currently used in clinical practice to inhibit neuronal overexcitation are mainly benzodiazepines. The present invention provides polyketide compounds that are completely different from the skeleton type of the currently used clinical drugs for inhibiting neuronal overexcitation. Based on these compounds, drugs for inhibiting neuronal overexcitation can be further developed to provide more choices for the market.

[0014] 2. The present invention is the first to reveal that the cage-structured polyketide compounds isolated from the fermentation products of Penicillium daleae have the activity of inhibiting neuronal excitability. In the future, the scientific community will focus on finding lead compounds with the activity of inhibiting neuronal excitability from Penicillium daleae.

[0015] 3. The cage-structured polyketone compounds to be protected by the present invention can be obtained by microbial fermentation, and the entire production process is free of chemical pollution and is green and environmentally friendly.

[0016] 4. The compounds of the present invention have the activity of significantly inhibiting the action potential discharge frequency and spontaneous excitatory post-stimulation current (sEPSCs) frequency of basket cell neurons in the hippocampus of mice, and are non-toxic to nerve cells N2a, and are useful for preparing new drugs for inhibiting neuronal overexcitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the hydrogen spectrum (600 MHz, CDCl3) of the compound of formula I prepared in a specific embodiment.

[0018] Figure 2 It is the carbon spectrum (150 MHz, CDCl3) of the compound of formula I prepared in a specific embodiment.

[0019] Figure 3 The figure shows the cytotoxicity of the compound of formula I at different concentrations in Example 1 to N2a cells.

[0020] Figure 4 This is a graph showing that the compound of formula I in Example 2 reduces the firing frequency of basket cells.

[0021] Figure 5 The frequency signal graph (AD left), cumulative probability graph (AD right) and statistical graph of sEPSC frequency change rate before and after administration of different concentrations of the compound of formula I in Example 2 inhibit spontaneous excitatory postsynaptic currents sEPSCs (ns, no significant difference; *P<0.05, statistically significant difference; **P<0.01, significant statistical difference; ***P<0.001, extremely significant statistical difference). DETAILED DESCRIPTION

[0022] In order to make the invention purpose and invention content of this application more clearly presented, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0023] In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are conventional commercially available products.

[0024] The animal experiments in the following examples have been approved by the Experimental Animal and Biomedical Ethics Committee of South-Central University for Nationalities (No. 2019-SCUEC-AEC-021).

[0025] The Penicillium daleae strain used was isolated from the root soil of Crystal Orchid, which was obtained from Tiantangzhai Scenic Area, Yingshan County, Hubei Province in August 2018. The strain has been deposited in the Medicinal Fungi and Ethnomedicine Research Group of the School of Pharmacy, South-Central University for Nationalities, numbered L3SO, and the address is: No. 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province, and the public can obtain the strain from this unit.

[0026] The cage-like polyketide compound studied in the present invention is derived from the fermentation product of Penicillium daleae. Its structure is shown in Formula I:

[0027]

[0028] The cage-like structure polyketone compound represented by formula I of the present invention is obtained as follows:

[0029] (1) Preparation of culture medium:

[0030] The rice culture medium formula is: in a 3L conical flask, add 400g rice and 400mL water respectively, sterilize at 121°C for 20 minutes and then cool at room temperature (room temperature in this application refers to 25°C). In this embodiment, 4kg of rice culture medium is prepared according to the above formula ratio.

[0031] (2) Fermentation

[0032] 1 mL of bacterial culture solution (about 1×10 8 After the spores were inoculated into rice culture medium, they were cultured at room temperature in a dark environment for 60 days to complete the fermentation.

[0033] (3) Extraction

[0034] (a) After fermentation, anhydrous methanol was used for immersion extraction (10 L each time, three days each time, a total of three times). After each immersion, the methanol extract was separated from the fermentation product using a centrifuge (Zhangjiagang Ding's Machinery), and the three extracts were combined. Then, a rotary evaporator, a vacuum pump and a low-temperature cooling circulation pump (i.e., a vacuum concentration device, the same below) were used to reduce the pressure and concentrate the methanol extract to remove the methanol to obtain a concentrate.

[0035] (b) The above concentrate was diluted with water to 2 L and then extracted with ethyl acetate in a 10 L separatory funnel in a volume ratio of 1:1 by shaking. After standing and stratification, the upper ethyl acetate layer was taken and the lower aqueous liquid was further extracted with ethyl acetate / water in a volume ratio of 1:1. After repeated extraction three times, the ethyl acetate layers were combined and concentrated under reduced pressure to remove ethyl acetate to obtain 80.5 g of crude extract.

[0036] (c) Add appropriate amount of methanol as solvent to the above crude extract to dissolve it, transfer it to a suitable evaporating dish, add 100g normal phase silica gel (80-100 mesh, Qingdao Ocean Chemical Co., Ltd.) to adsorb the sample, evaporate the solvent in a water bath (constant temperature 50°C) and grind it into powder. Take a glass column, add 300g new normal phase silica gel and the above powder in turn, and use petroleum ether and acetone gradient elution (in order: pure petroleum ether elution 2L, volume ratio 20:1 elution 2L, volume ratio 15:1 elution 2L, volume ratio 10:1 elution 2L, volume ratio 5:1 elution 2L, volume ratio 2:1 elution 2L, pure acetone elution 1L), every 200 ml is transferred to a bottle, concentrated under reduced pressure until the solvent is evaporated to dryness, dissolved with a small amount of methanol, and transferred to a 10mL penicillin bottle with a rubber dropper. The samples from all the vials were sampled with a capillary and spotted on a silica gel plate G254 (Qingdao Ocean Chemical Co., Ltd.), and developed in a 200-mL developing cylinder with 5 mL of a mixed solvent of petroleum ether and acetone (volume ratio of 3:2). After the solvent reached the top, it was taken out and dried, and the fluorescence of the compound was observed under an ultraviolet lamp (254 nm). The silica gel plate was evenly sprayed with vanillin-concentrated sulfuric acid-ethanol solution, and heated on an electric furnace for color development. The same or similar components were combined, and the elution component at a volume ratio of petroleum ether to acetone of 5:1 was collected and recorded as component E.

[0037] (d) Component E was dissolved in a small amount of methanol, filtered through cotton and added to a dextran gel chromatography column (LH-20). The eluent was pure methanol. One tube was connected for every 10 mL. TLC detection was performed (the developing solvent was a mixed solvent of petroleum ether and acetone in a volume ratio of 3:2). The fluorescent spot under 254 nm ultraviolet light was used as the main reference, and vanillin-concentrated sulfuric acid-ethanol solution was used for color development. f The components of the spot with a size of ≈0.5-0.6 were combined and evaporated to dryness under reduced pressure, and recorded as component E1. Component E1 was dissolved in a small amount of chromatographic grade methanol, filtered into a 1.5 mL liquid phase vial using a 0.22 μm filter head, and separated and purified on a preparative high performance liquid chromatography (Agilent 1260, USA) to obtain the compound of formula I. Conditions: Zorbax SB-C 18 Column (particle size 5 μm, size 9.4×150 mm, flow rate 4 mL·min -1 ), the mobile phase was acetonitrile and water (v / v, 35:65 to 50:50, 30 min), the detector was DAD (photodiode array detector), the detection wavelengths were 205, 220, 254, 280 and 365 nm, and the peak at the retention time of 27 minutes was the compound of formula I.

[0038] Structure identification:

[0039] Purify the obtained compound: The solvent was concentrated and evaporated to dryness under reduced pressure, dissolved in 0.45 mL of deuterated chloroform, and then transferred to a nuclear magnetic resonance tube. The hydrogen spectrum and carbon spectrum ( Figure 1 , Figure 2 ) and two-dimensional spectra, HR-ESI-MS confirmed that its molecular formula is C 31 H 40 O 10 (m / z 573.26941[M+H] + , the calculated value is C 31 H 41 O 10 ,573.26997) has a total of 12 degrees of unsaturation, and the final confirmed structural formula is shown in Formula I, which belongs to a cage-structured polyketide compound.

[0040] The cage-structured polyketone compound represented by formula I of the present invention is soluble in solvents such as dichloromethane, chloroform, acetone, methanol, pyridine, dimethyl sulfoxide, etc., but is insoluble in water.

[0041] Example 1

[0042] The CCK-8 method was used to detect the cytotoxicity of cage-structured polyketide compounds on neuronal cells N2a (Neuro-2a, purchased from Wuhan Sean Biotechnology Co., Ltd.).

[0043] Experimental principle: After CCK-8 solution (active ingredient is WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) is added to the cell suspension, WST-8 is oxidized and reduced by intracellular dehydrogenase in the presence of electron carriers to generate orange-yellow formazan dye, which can be dissolved in tissue culture medium, and the amount of formazan generated is proportional to the number of living cells.

[0044] N2a cells were inoculated into Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% fetal bovine serum and 1% penicillin / streptomycin and activated and cultured at 37°C and 5% CO2. In order to maintain the good condition of the cells, the medium was replaced periodically, and the cells were passaged at a density of 80% using 0.25% trypsin. The cultured N2a cell suspension was plated at 7×10 3 Cells / well were inoculated in a 96-well plate, and the compound of formula I prepared by DMSO gradient dilution method was added to make the final concentrations 0.5, 1, 10, and 20 μM, respectively. DMEM was added to make the final volume of each well 200 μL. A negative control group (i.e., the following 0 加药The cells were grown in the same group (group 1, adding an equal amount of DMSO and supplementing DMEM medium to a final volume of 200 μL per well), and then grown at 37°C and 5% CO2 for 24 hours. Then, 10 μL of CCK-8 solution was added to each well and incubated for another hour at 37°C in the dark. Finally, the absorbance of each well was measured at 450 nm using a 96-well microplate reader (Thermo Fisher Scientific, USA), and the cell viability was calculated.

[0045] Cell viability calculation:

[0046] Cell viability*(%)=[A (加药) -A (空白) ] / [A (0加药) -A (空白) ]×100%;

[0047] A (加药) : absorbance of wells with culture medium, cells, CCK-8 solution, and drug solution;

[0048] A (空白) : absorbance of wells with culture medium and CCK-8 solution but without cells;

[0049] A (0加药) : absorbance of wells with culture medium, cells, CCK-8 solution but no drug solution;

[0050] *Cell viability: cell proliferation activity or cytotoxic activity, in this example Figure 3 The cell viability is shown in the table.

[0051] The test results are as follows Figure 3 As shown, the compound of formula I showed no cytotoxicity to the neuronal cells N2a at a concentration of 500 nM-20 μM.

[0052] Example 2

[0053] The patch clamp technique was used to detect the inhibitory activity of the cage-structured polyketide compounds on the action potential discharge frequency and spontaneous excitatory post-synaptic current (sEPSCs) frequency of male rat hippocampal basket cell neurons.

[0054] Experimental principle: The patch clamp technique uses a glass microelectrode to contact the cell membrane and connect them with an impedance of more than a gigaohm. The small area of ​​the cell membrane (membrane patch) connected at the opening of the microelectrode tip is electrically separated from its surroundings. On this basis, the potential is fixed, and the ionic current (pA level) of the ion channel on this membrane patch is detected and recorded.

[0055] The specific steps of the patch clamp technique detection in this embodiment are as follows: All male mice were anesthetized with 2% isofuran and then quickly decapitated. The brain tissue after preliminary trimming was immersed in an oxygen-saturated slicing solution (185mM sucrose, 2.5mM KCl, 2mM MgSO4, 2mM CaCl2, 26mM NaHCO3, 1.2mM NaH2PO4, 25mM glucose, pH7.4) ice-water mixture. The coronal sections were cut with an oscillating microtome (Leica VT1200S), and the slice thickness was 300μm. After cutting, the brain slices were placed in an incubator containing oxygen-saturated ACSF (artificial cerebrospinal fluid), which contained 124 mM NaCl, 1.25 mM NaH2PO4, 2.5 mM KCl, 1.3 mM MgCl2, 0.5 mM CaCl2, 26 mM NaHCO3, 10 mM glucose, pH 7.3-7.4, and incubated at 32-34°C for half an hour, the temperature being controlled by a TC-324C dual automatic temperature controller (Warner Instruments, USA), and bubbling continuously under the action of 5% CO2 and 95% O2. After the incubation, the slices were transferred to 25°C to start the experiment, and the data were recorded.

[0056] First, find suitable neurons with good activity under an infrared differential interference microscope (Olympus, Japan), fill the microelectrode (3-8MΩ) with electrode solution (122mM potassium gluconate, 5mM NaCl, 2mM MgCl2, 10mM HEPES, 1mM EDTA, 10mM sodium creatine phosphate, 4mM Na2ATP, 0.4mM Na3GTP, pH 7.3), use a micromanipulator to manipulate the microelectrode to approach the neuron in BATH mode, when the resistance increases by 0.3-0.5MΩ, remove the positive pressure, and use appropriate negative pressure to make the cell membrane adsorb on the opening of the electrode tip, when the sealing resistance is higher than 1GΩ, remove the negative pressure, and a high-resistance seal is formed. Compensate for the cell fast capacitance as needed, continue to apply appropriate negative pressure until the cell membrane at the seal is broken, switch to patch mode, observe that the resistance remains at or above 1GΩ, and switch to whole cell mode for subsequent electrophysiological detection and recording:

[0057] (1) Depolarize the neuron with a 10pA gradient from 10 to 300pA for 1000ms, and then stimulate the membrane with a 90-110pA current. Monitor and record the changes in action potential frequency before administration (final concentration 5μM, prepared by adding the mother solution of Formula I dissolved in DMSO to the electrode solution and diluting, circulatory perfusion administration for 15min), and after washing away the drug with the electrode solution. Two-way ANOVA was used to analyze the experimental results. P<0.05 was considered statistically significant (the experimental results are shown in Table 1). Figure 4 shown).

[0058] (2) 10 μM bicuculline (dissolved in DMSO) was added to ACSF to block GABA receptors. The signal was filtered at 2 kHz and sampled at 20 kHz through an Axon 700B amplifier and an Axon 440A analog / digital interface (MeiGu Molecular Instruments, USA). Neurons were depolarized by stimulating with a 10pA gradient from 10 to 300pA for 1000ms, and spontaneous excitatory postsynaptic currents were recorded in the gap-free mode. All neurons were maintained at -70 mV in the voltage clamp mode. The membrane patches were divided into 0.5 μM, 1 μM, 10 μM, and 20 μM drug concentration groups (all obtained by dissolving the compound of formula I in DMSO) and a control group (DMSO group). The spontaneous excitatory postsynaptic currents were recorded before drug addition, and then the drug was added. After waiting for 3-5 minutes, the spontaneous excitatory postsynaptic currents were recorded again. The experimental data were statistically analyzed for differences between the groups using one-way analysis of variance. P < 0.05 was considered statistically significant (the experimental results are shown in Figure 2). Figure 5 shown).

[0059] The test results are as follows Figure 4 As shown, the compound of formula I at a concentration of 5 μM significantly reduced the discharge frequency of blue cells under 90-110 pA current stimulation. Figure 5 As shown, the compound of formula I can significantly reduce the frequency of spontaneous excitatory postsynaptic current (sEPSC) of blue cells at the used concentrations (0.5 μM, 1 μM, 10 μM, 20 μM). At a concentration of 20 μM, the frequency is reduced by nearly half, and the frequency of sEPSC is concentration-dependent with the dosage of the compound of formula I.

[0060] In summary, the cage-structured polyketide compounds isolated from the fermentation extract of Penicillium daleae provided by the present invention have significant activity in inhibiting neuronal excitability and can be used as candidate molecules of lead compounds with activity in inhibiting neuronal excitability for preparing drugs that inhibit excessive neuronal excitability.

[0061] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. Use of a special cage-structured polyketide compound in the preparation of a drug capable of inhibiting neuronal overexcitation, characterized in that: The special cage-structured polyketone compound is a compound having a chemical structural formula as follows:

2. The use according to claim 1, characterized in that: The compound of formula I is used in combination with or without other active ingredients when preparing drugs for inhibiting neuronal overexcitation.

3. The use according to claim 2, characterized in that: The special cage-structured polyketone compound is the only active ingredient.

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