Compound stereum alkyd A co-cultured by medicinal fungi stereum hirsutum and stereum giganteum as well as preparation method and application of compound

Abstract

The invention relates to the technical field of medicinal compounds, and particularly discloses a compound separated from medicinal fungus stereum hirsutum and stereum chroocoides co-culture fermentation liquor as well as a preparation method and medical application of the compound. The compound is named as Vibrahirsutin A, and the compound is named as Vibrahirsutin A. The invention further discloses a preparation method of the compound. The compound disclosed by the invention has relatively remarkable neuroactivity, can be used for preparing new neuroprotective drugs and analgesic drugs, and is used for treating cognitive impairment and motion retardation including but not limited to improvement of neurodegenerative diseases and relieving pain. Results of the invention show that the stereic acid A can significantly increase the current frequency after spontaneous excitatory synapses of neurons, has a half maximum effective concentration of 23.96 + / -0.016 mM, and has a good analgesic effect on chronic migraine model mice stimulated by inflammatory decoction.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal compounds, and in particular to a compound oleic acid A extracted from the co-culture fermentation of medicinal fungi Leptotrichum pubescens and Leptotrichum fusca, a preparation method thereof, and application thereof in the pharmaceutical field. Background Art

[0002] Stereum hirsutum is a wood-rotting fungus belonging to the genus Stereum, Basidiomycotina, Basidiomycetes, Russulales, Stereaceae, Eumycota. Stereum hirsutum is rich in terpene synthase, and more than 40% of the secondary metabolites isolated from the fungus are sesquiterpenoids, which have antibacterial, anti-inflammatory, antioxidant, anticancer, and cytotoxic activities. The remaining compound types include styrosin derivatives, sterols, aromatic alkynes, benzoates, etc. In addition to the above activities, pancreatic lipase inhibition activity, α-glucosidase inhibition activity, glyoxalase inhibition activity, and phytotoxic activity were also detected (Zhao Zhenzhu, Study on the chemical composition and biological activities of six fungi such as Matsutake, PhD dissertation of University of Chinese Academy of Sciences, 2018).

[0003] Boreostereum vibrans is a fungus of the same genus as Stereum. Vibralactone isolated from its fermentation broth has a rare β-lactone 4 / 5 fused bicyclic skeleton and significant pancreatic lipase inhibitory activity (Organic Letters, 2006, 8, 25, 5749–5752). Most of the compounds isolated from Boreostereum vibrans are derivatives of vibralactone, and there are also some sesquiterpenes and a few other types of compounds (Natural Products and Bioprospecting 2014, 4, 271–276; Natural Products and Bioprospecting 2018, 8, 37–45; Natural Products and Bioprospecting 2012, 2, 200–205).

[0004] Both fungi can produce tenascin derivatives and sesquiterpenes, but the yields are very different. Tenascin produces more sesquiterpenes, while Tenascin mainly produces tenascin derivatives. Currently, both are studied separately, and there are few reports on the co-cultivation of the two fungi. Therefore, it is of great significance to fully study the secondary metabolites and pharmacological activities of the medicinal fungi Tenascin and Tenascin co-cultivated.

[0005] Neurodegenerative diseases adversely affect the lives of tens of millions of people worldwide and lead to the progressive loss of neurons in the central nervous system or peripheral nerves, ultimately causing memory loss, cognitive decline, and impairment of behavior, sensory and / or motor function. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and amyotrophic lateral sclerosis.

[0006] Pain is a common clinical symptom. With the increasing aging of society, pain has become the third largest health problem after cardiovascular and cerebrovascular diseases and tumors. Pain patients not only suffer physical pain, but also suffer great mental effects, often accompanied by anxiety and depression, which seriously affects the quality of life. Before symptomatic treatment, analgesics are usually used to relieve pain and improve the condition. Migraine is a common type of primary headache in clinical practice, with moderate to severe throbbing headaches, mostly unilateral. The global prevalence of migraine is about 11.6%, which seriously affects the physical and mental health of patients, and imposes a huge burden on patients' families and society. It has become one of the major public health problems in the world (Neurological Diseases and Mental Health, 2024, 24(03):223-228; Chinese Herbal Medicine, 2024, 55(13):4445-4454).

[0007] Therefore, the market development potential of drugs for treating neurodegenerative diseases and migraine is considerable. Summary of the invention

[0008] In view of the problems and shortcomings in the above-mentioned prior art, the object of the present invention is to provide a compound obtained by co-culturing the medicinal fungi Lederma lucidum and Lederma fusca, and to study its activity.

[0009] The inventors of the present application have systematically studied the chemical components of the co-cultured fermentation products of Leptotrichum pubescens and Leptotrichum chinense, and discovered the compound Leptotrichum suis A of formula (1). Studies on the neuropharmacological activity of Leptotrichum suis A have shown that the molecule can significantly promote the occurrence of action potentials in pyramidal neurons of the insular cortex of mice, increase the excitability of neurons, and enhance the frequency of spontaneous excitatory postsynaptic currents of the same type of neurons, suggesting that the compound has application prospects in the development of drugs for the treatment of neurodegenerative diseases. In addition, Leptotrichum suis A also has a significant analgesic effect on chronic migraine, and can be used to prepare analgesics, preferably analgesics for the treatment of chronic migraine.

[0010] To achieve the above-mentioned object of the present invention, the present invention provides a compound obtained by co-culturing medicinal fungi Lederma pubescens and Lederma fusca: 12 H 18 O5, chemical name is tough leather alkyd A, structural formula is as follows (1):

[0011]

[0012] Its stereo configuration is shown in formula (2):

[0013]

[0014] The present invention also provides a method for extracting the novel compound represented by the above formula (1), the steps of which are as follows:

[0015] (1) inoculating the activated Lederma lucidum strain and the Lederma lucidum strain in a potato-glucose liquid medium at a dosage ratio of 0.1 to 1:1, co-culturing and fermenting for a certain period of time under dark conditions, filtering and separating the mycelium and the fermentation liquid, soaking the mycelium in a solvent, mixing the soaking liquid with the fermentation liquid, and concentrating under reduced pressure to obtain a crude extract;

[0016] (2) dissolving the crude extract in water, extracting with ethyl acetate, and concentrating under reduced pressure to remove the ethyl acetate to obtain an extract;

[0017] (3) The extract obtained in step (2) was separated by normal phase silica gel column chromatography, using petroleum ether and acetone as eluents, eluting in a gradient of 20:0, 20:1, 1:1, and 0:1, v / v, and collecting the 1:1 elution fraction;

[0018] (4) The eluted fractions collected in step (3) were separated by medium pressure liquid chromatography using reverse phase silica gel, with methanol and water as the eluent, and eluted in a gradient of 20:80, 40:60, 60:40, 80:20, and 100:0, v / v, to obtain a total of 12 fractions, and the eluate with a methanol to water volume ratio of 40:60 was collected to obtain component B;

[0019] (5) Component B was eluted by Sephadex LH-20 gel column chromatography, the eluent was pure acetone, and TLC was used for detection. The fluorescent spot under 254nm ultraviolet light was used as a reference, and vanillin-concentrated sulfuric acid-ethanol solution was used for color development. The fractions were combined according to the color spots to obtain two components, B1 and B2, of which the main spot of B2 was purple-red, R f The value is about 0.5 component, and the B2 component is passed through a normal phase chromatography column. The eluent is petroleum ether and acetone. The elution is carried out in a gradient of 6:1, 4:1, 2:1, 1:1, 0:1, v / v, and the components eluted when the volume ratio of petroleum ether to acetone is 4:1 are collected. The components are washed with petroleum ether and the petroleum ether is removed to obtain tough leather alcohol acid A.

[0020] Furthermore, the potato-glucose liquid culture medium is a modified potato liquid culture medium, which contains, per liter, a cooking liquid prepared from 200 g of peeled potatoes, 20 g of glucose, 1 g of peptone, 1.5 g of KH2PO4, 1.5 g of MgSO4, and 10 mg of vitamin B1.

[0021] Furthermore, the D. pubescens strain and the D. chinensis strain are both activated in a modified potato-glucose-agar medium (obtained by adding 17 g agar per liter of the modified potato liquid medium), and the preferred activation condition is: culturing at 25° C. for 7 days.

[0022] Furthermore, in step (1), equal amounts of the two activated bacterial beads (0.25 mm 2 The mycelium-containing agar block) was inoculated into the prepared culture medium and cultured at a constant temperature of 25° C. in a dark environment for 25 days to complete the fermentation; the solvent was a mixed solvent of chloroform and methanol in a volume ratio of 1:1, and the immersion extraction was performed 5 times, each time for 24 hours.

[0023] The present invention also discloses the use of the compound oleic acid A or a pharmaceutically acceptable preparation thereof in the preparation of a drug for treating neurodegenerative diseases and / or a drug for treating pain.

[0024] Furthermore, the drug for treating neurodegenerative diseases is a drug for treating Alzheimer's disease, Parkinson's disease, Huntington's disease and / or amyotrophic lateral sclerosis.

[0025] Furthermore, the drug for treating pain is a drug for treating chronic migraine.

[0026] In the present invention, the neuropharmacological activity of the compound of formula (1), oleic acid A, is specifically embodied in that it can enhance the spontaneous excitatory postsynaptic current (sEPSC) frequency of the same type of neurons within a concentration range of 1-100 μM, and the activity is concentration-dependent with the administration concentration of the compound, and its half maximum effective concentration (EC 50 ) value was 23.96±0.016μM, and the amplitude of neuronal sEPSC did not change before and after administration. In addition, the compound tenascin A can affect the excitability of pyramidal neurons in the insular cortex of mice, specifically, the compound tenascin A has a promoting effect on the frequency of neuronal action potentials. Therefore, the tenascin A has a potential use in preparing new anti-neurodegenerative disease drugs.

[0027] The compound oleic acid A also has a significant analgesic effect on chronic migraine (CM) model mice stimulated by inflammatory soup. The compound oleic acid A can significantly increase the acute mechanical withdrawal threshold and thermal withdrawal latency of the hind paw and periorbital area of ​​mice. Therefore, the oleic acid A can effectively reduce the pain response tendency of CM mice and can be used to prepare analgesics, preferably for the preparation of analgesics for chronic migraine.

[0028] The above technical solution of the present invention was completed with the support of the following funds: National Natural Science Foundation of China (No.82473810) and the Central Universities Basic Research Business Expenses Special Funds Project (No.CPT22033).

[0029] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0030] 1. The medicinal fungi of the present invention, Lederma lucidum and Lederma fusogenum, are co-cultured to produce a natural compound with a completely new skeleton, which is completely different from the chemical structure of any single-ingredient drug currently on the market. Based on this compound, further development into a drug specifically for the treatment of neurodegenerative diseases and analgesia can avoid the patents of existing marketed drugs and provide more options for the market.

[0031] 2. The present invention reveals for the first time that the derivatives of tenascin (the biogenic precursor compound of tenascin alcohol A is tenascin) have significant neuropharmacological activity, which will help the field of drug research and development to focus on the neuropharmacological activity of the secondary metabolites of tenascin and to find more new natural molecules that act on the activity of the nervous system.

[0032] 3. The tough leather alcohol acid A to be protected in the present invention is abundant in source and can be continuously obtained through microbial fermentation. The entire production process is chemical-free and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the HRESIMS spectrum of the new compound obtained in Example 1 of the present invention;

[0034] Figure 2 The new compound obtained in Example 1 of the present invention 1 H-NMR spectrum;

[0035] Figure 3 The new compound obtained in Example 1 of the present invention 13 C-NMR spectrum;

[0036] Figure 4 DEPT135 NMR spectrum of the new compound obtained in Example 1 of the present invention;

[0037] Figure 5 This is the nuclear magnetic resonance HSQC spectrum of the new compound obtained in Example 1 of the present invention;

[0038] Figure 6 The HMBC nuclear magnetic resonance spectrum of the new compound obtained in Example 1 of the present invention is shown in FIG.

[0039] Figure 7 The NMR of the new compound obtained in Example 1 of the present invention is 1 H- 1 H COSY spectrum;

[0040] Figure 8 The ROESY nuclear magnetic resonance spectrum of the new compound obtained in Example 1 of the present invention is shown in FIG.

[0041] Fig. 9 This is the X-ray single crystal diffraction pattern of the new compound obtained in Example 1 of the present invention;

[0042] Fig.10 The effect of compound 1 at a concentration of 1-100 μM on the activity of Neuro2A in Example 2;

[0043] Fig.11 The effect of compound 1 in Example 2 on the action potential of pyramidal neurons in the insular cortex;

[0044] Fig.12 and Fig.13 They are the effect of compound 1 in Example 2 on sEPSC of pyramidal cells in the insular cortex and the dose-effect curve of compound 1;

[0045] Fig.14 This is the improving effect of compound 1 in Example 2 on chronic migraine behavior in mice. DETAILED DESCRIPTION

[0046] The applicant will now provide a clear and complete description of the technical solution of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings.

[0047] In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional commercial products. The materials and reagents used, unless otherwise specified, are all common commercial reagents with analytical purity.

[0048] 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).

[0049] In the following embodiments: Stereum hirsutum was collected in Jianchuan County, Yunnan Province, and was identified by ITS gene sequencing (ITS sequence submitted to GenBank, accession number is No. MG655586), the strain number is HFG27, and it is also the strain used in the document "Stereuins A–F: Isopentenyl benzene congeners with antibacterial and neurotrophic activities from Stereum hirsutum HFG27" (Zhen-Zhu Zhao et al, Phytochemistry, 228, 114253, 2024, DOI: 10.1016 / j.phytochem.2024.114253), the strain is stored in the Ethnic Medicine and Medicinal Fungi Research Group, School of Pharmacy, South-Central University for Nationalities, the address is: No. 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province, and the public can purchase the strain from the unit.

[0050] Borotereum vibrans was collected from Kunming Botanical Garden, Yunnan, and is also the strain used in the literature "Novel Natural Oximes and Oxime Esters with a Vibralactone Backbone from the Basidiomycete Boreostereum vibrans" (He-Ping Chen et al, Chemistry Open, 5, 142-149, 2016, 10.1002 / open.201500198). The strain is currently in the National Medicine and Medicinal Fungi Research Group of South-Central University for Nationalities, No. 20120920B, and the address is: No. 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province. The public can purchase the strain from this unit.

[0051] Example 1: Preparation of the compound of formula (1)

[0052] Step 1: Prepare 250 bottles of 400 mL modified potato liquid culture medium (each liter contains boiling water prepared from 200 g peeled potatoes, 20 g glucose, 1 g peptone, 1.5 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, the same below), and sterilize it at 121°C for 25 minutes. Then inoculate S. hirsutum and B. vibrans (0.5 mm each) 2 The agar blocks containing mycelium) were activated on a potato-glucose-agar medium (prepared by adding 17 g agar per liter of the above-mentioned improved potato liquid medium), and cultured at 25°C for 7 days. When the mycelium grew to about 1 cm from the edge of the plate, 0.5 mm×0.5 mm agar blocks containing mycelium were dug out and inoculated into the prepared improved potato liquid medium. The fermentation was completed by constant temperature culture at 25°C in a dark environment for 25 days;

[0053] Step 2: Filter the 100L fermentation product to separate the fermentation broth and mycelium, soak the mycelium in a mixed solvent of chloroform and methanol in a volume ratio of 1:1 for 5 times, each time for 24 hours, filter after each soaking, combine the filtrate obtained 5 times and mix it with the fermentation broth, and concentrate under reduced pressure to obtain a crude extract. Dissolve the crude extract in 2.5L of water and then extract it with 5L of ethyl acetate in a 10L separatory funnel. After standing for 2 hours after shaking, collect the ethyl acetate layer, and continue to extract the lower water layer with 5L of ethyl acetate. After combining the collected ethyl acetate layers, use a rotary evaporator to concentrate to obtain 36.33g of extract;

[0054] Step 3, separating the obtained extract by using 80-100 mesh normal phase silica gel column chromatography, using petroleum ether-acetone as eluent, eluting in a gradient of 20:0, 20:1, 1:1, 0:1, v / v, and collecting the eluate with a volume ratio of petroleum ether to acetone of 1:1;

[0055] Step 4: The eluate obtained in step 3 was separated by reverse phase silica gel column chromatography (medium pressure liquid chromatography was used in the experiment), and the stationary phase was C 18 Silica gel (YMC ODS-A-HG, product number ODS-A-HG), column length 50 cm, column inner diameter 6 cm, gradient elution with methanol and water, volume ratio of methanol to water is 20:80, 40:60, 60:40, 80:20, 100:0, each gradient elution 1L, collect the eluate with volume ratio of methanol to water of 40:60 to obtain component B;

[0056] Step 5: Component B obtained in step 4 was eluted by Sephadex LH-20 gel column (acetone) chromatography, the eluent was pure acetone, and TLC detection was adopted (the developing solvent was a mixed solvent of petroleum ether and acetone in a volume ratio of 3:2), the fluorescent spot under 254nm ultraviolet lamp was used as the main reference, and vanillin-concentrated sulfuric acid-ethanol solution was used for color development, and the fractions were merged according to the color spots to obtain two components, B1 and B2, wherein the main spot of component B1 was light blue, and R f The value is about 0.76, the main point of B2 component is purple, R f The value is about 0.5. Component B2 was further subjected to a normal phase chromatography column (petroleum ether-acetone, volume ratio 6:1, 4:1, 2:1, 1:1, 0:1) to obtain five sub-components, among which component B2-2 was collected when the volume ratio of petroleum ether to acetone was 4:1, and the TLC detection and color development showed a single purple-red spot. After merging, the organic solvent was evaporated to obtain a colorless prismatic solid with a slightly yellow surface. The yellow substance on the surface was quickly washed with petroleum ether to obtain a crystalline compound 1 (47.5 mg), named as tough leather alcohol acid A.

[0057] Structural identification: Using modern spectroscopic techniques such as 1 H NMR spectroscopy, 13 The new compound obtained in step 5 was structurally identified by C NMR, DEPT 135 NMR, 2D NMR (HSQC, HMBC), and high resolution mass spectrometry (HRESIMS). Figure 1-8 ;

[0058] The molecular formula was determined to be C by high resolution mass spectrometry (HRESIMS). 12 H 18 O5(m / z 243.12277[M+H] + , the calculated value is C 12 H 19 O5, 243.12270), unsaturation is 4; 1 H(600MHz), 13 The C (150 MHz) spectrum data are shown in Table 1.

[0059] Table 1: Compound 1 1 H(600MHz), 13 C (150MHz) spectral data (deuterated methanol)

[0060]

[0061] The results of 2D NMR, HMBC and COSY spectra of the obtained compound are as follows:

[0062]

[0063] Then, the planar configuration of the obtained compound was determined by 2D NMR: starting from the methyl signal, the H3-11 / 12 and C-10 (δ C 82.3), C-9(δ C 54.1) are related, H-9 and C-2 (δ C 82.4), C-3(δ C 61.7), C-4(δ C 38.4) are related, H2-8 and C-7 (δ C 176.8), C-2(δ C 82.4), C-5(δ C 72.1), C-1(δ C 61.4) are related, H2-4 and C-2 (δ C 82.4), C-13(δ C 66.0), C-3(δ C 61.7) are related, H2-13 and C-2 (δ C 82.4), C-3(δ C 61.7), C-9(δ C 54.1). COSY spectrum showed the correlation of H2-4 / H-5 and H2-8 / H-9. Combined with HRESIMS results, the planar structure of the obtained compound was confirmed. Fig. 9 ), verified the correctness of the planar structure analysis of compound 1 and determined the absolute configuration of compound 1.

[0064] The structure of the resulting compound is shown below:

[0065]

[0066] Example 2: Study on the neuropharmacological activity of compound 1 obtained in Example 1

[0067] Experiment 1: CCK-8 method was used to detect the cytotoxicity of compound 1 on nerve cells N2a:

[0068] The CCK-8 assay was used to determine whether the test samples at a concentration ranging from 1 to 100 μM had cytotoxic activity against the growth of mouse neuroblastoma cells (Neuro2A, purchased from Wuhan Sean Biotechnology Co., Ltd.) within 24 hours.

[0069] N2a (Neuro-2a) was added at a rate of about 5 × 10 3The cells were inoculated into a 96-well plate containing Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the volume of the medium was 100 μL / well. The drug solution of the compound of formula (1) prepared by the DMSO gradient dilution method was added to the above 96-well plate to make the final concentrations of 1, 10, 30, 50, and 100 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 460 nm using a 96-well microplate reader (Thermo Fisher Scientific, USA), and the cell viability was calculated.

[0070] Cell viability calculation:

[0071] Cell viability*(%)=[A (加药) -A (空白) ] / [A (0加药) -A (空白) ]×100%;

[0072] A (加药) : absorbance of wells with culture medium, cells, CCK-8 solution, and drug solution;

[0073] A (空白) : absorbance of wells with culture medium and CCK-8 solution but without cells;

[0074] A (0加药) : absorbance of wells with culture medium, cells, CCK-8 solution but no drug solution;

[0075] *Cell viability: cell proliferation activity or cytotoxic activity, in this example Fig.10 The cell viability is shown in the table.

[0076] The test results are as follows Fig.10 As shown, the compound of formula (1) showed no cytotoxicity to the nerve cells N2a at a concentration of 1-100 μM, which verified the safety of the administration of compound 1.

[0077] The patch clamp technique was used to detect the inhibitory activity of the compound of formula (1) on the action potential discharge frequency and spontaneous excitatory post-synaptic current (sEPSCs) frequency of male rat hippocampal basket cell neurons.

[0078] Experimental animals used in the following experiments: Male C57BL / 6 mice (20-25 g) were housed under SPF (Specific Pathogen Free) conditions with an ambient temperature of 25 ± 1 °C, a humidity of 60 ± 2%, a 12-hour dark / light cycle, and unlimited access to food and water. All animal experiments were carried out in strict accordance with the guidelines for animal experiments, and the use and care of animals were approved by the Animal Ethics Committee of South-Central University for Nationalities (SYXK (Wuhan) 2016-0089 No. 2021-SCUEC-AEC-033).

[0079] Experiment 2: Effects on the action potential of pyramidal neurons in the insular cortex:

[0080] 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.

[0081] 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.

[0082] First, find 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:

[0083] The neuron was depolarized by stimulating with a 10pA gradient from 10 to 300pA for 1000ms, and then the membrane was stimulated with a 50-70pA current. The action potential frequency changes were monitored and recorded before administration (final concentration 10μM, obtained by adding the mother solution of the compound of formula (1) dissolved in DMSO to the electrode solution and diluting, and circulatory perfusion administration for 15min), and after the drug was washed away with the electrode solution. The experimental results were analyzed by two-way ANOVA, and P<0.05 was considered statistically significant.

[0084] Fig.11 It can be observed that after perfusion of compound 1, the frequency of neuronal action potential discharge is significantly accelerated, which shows that compound 1 can promote the frequency of neuronal action potential discharge.

[0085] Experiment 3: Effects on sEPSC of pyramidal neurons in the insular cortex of mice:

[0086] On the basis of Experiment 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 by Axon 700B amplifier and Axon440A analog / digital interface (Megu Molecular Instruments, USA). Neurons were depolarized by stimulating with a 10pA gradient from 10 to 300pA for 1000ms, and spontaneous excitatory post-synaptic currents were recorded in Gap-free mode. All neurons were maintained at -70 mV in voltage clamp mode. The membrane patches were divided into 1 μM, 10 μM, 30 μM, 50 μM, 100 μM dosing concentration groups (all obtained by dissolving the compound of formula (1) in DMSO) and control group (DMSO group). The spontaneous excitatory post-synaptic current (sEPSC) before drug addition was recorded, and then the drug was added, and the spontaneous excitatory post-synaptic current was recorded again after waiting for 3-5 minutes. The experimental data were analyzed by one-way analysis of variance for the differences among the groups, and P < 0.05 was considered statistically significant.

[0087] The results are as follows Fig.12 As shown in the figure, the amplitude of neuronal sEPSC did not change significantly, but its frequency increased significantly with the increase of drug concentration, indicating that the effect of compound 1 on neuronal sEPSC is determined by presynaptic factors, and its half effective concentration (EC 50 ) value was 23.96±0.016μM (calculated based on the concentration and action intensity, using Graphpad software, such as Fig.13 shown).

[0088] Experiment 4: Study on the effect of treating chronic migraine:

[0089] Chronic migraine (CM) often occurs in middle-aged people, affecting the health and normal life of the patients, and causing a heavy economic burden on both individuals and society. Drugs for treating chronic migraine include analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), triptans, etc., and preventive drugs such as calcitoningene-related peptide (CGRP) have been developed in recent years. However, the treatment of chronic migraine is a long process. On the one hand, it is necessary to focus on the development of therapeutic drugs, and on the other hand, it is necessary to study whether it can effectively prevent or reverse the structural and functional lesions caused by the disease.

[0090] In this embodiment, the inflammatory soup model was used: a small hole was drilled in the back of the mouse skull and an internal tube was introduced into it. After the mouse was out of anesthesia, the inflammatory soup (1mM bradykinin, 1mM 5-hydroxytryptamine agonist, 1mM histamine and phosphate buffer solution containing 0.1mM prostaglandin E2, the phosphate buffer solution used was 0.1M, pH=7.4) was injected from the internal tube, 10μL each time, once every two days, about 5 days (the third injection of inflammatory soup), when the mouse hind paws touched the hot plate and immediately retracted the head when the front paws on the same side scratched the face, the model was considered successful. The experimental group was administered compound 1 (prepared with physiological saline to a 1.4μg / μL stock solution) to the inflammatory soup model mice, once every two days, and 14μg was administered to each mouse each time. The experiment set up a chemical drug positive control group (Control, administered with zolmitriptan tablets, dissolved in saline, 10 μL per dose at a concentration of 0.00405 g / kg) and a model group (Model, administered with 10 μL saline each time), and the administration frequency was once every two days. After administration, the following experiments were performed to evaluate the therapeutic effect of the sample on chronic migraine, specifically: the periorbital mechanical pain threshold and hind paw mechanical pain threshold of mice were measured using an electronic Von Frey mouse pain threshold meter (IITC Life Science Instruments, CA, USA). For the periorbital mechanical pain threshold measurement, the pain response was manifested as the tested mice avoiding, shaking their heads, or using their front paws to push away the pain meter; for the hind paw mechanical pain threshold measurement, the pain response was manifested as the tested mice shrinking their feet and licking their feet; a plantar heat tingling instrument (SA709, Jiangsu Saiangsi Biotechnology Co., Ltd.) was used to apply controllable temperature heat stimulation to the soles of the mice, and the latency of lifting the foot was recorded. The experiment was measured 7 times in a row for 13 days.

[0091] The experimental results are shown in Fig.14 Compared with the model group, the positive drug control group and the compound 1 administration group showed significant differences. Compound 1 has a significant improvement effect on the acute mechanical withdrawal threshold of the mouse hind paw and periorbital area, and also significantly increases the mechanical pain range of the mouse hind paw, so it has the effect of improving chronic migraine behavior. It is worth noting that when the dosage reaches 22μg, the mice will develop epileptic symptoms after 3-4 days, and will die the next day after the symptoms appear. After exploration, 14μg is the maximum dosage to ensure that the mice do not develop epileptic symptoms.

Claims

1. A compound obtained by co-culturing the medicinal fungi Lederma pubescens and Lederma fusca: the chemical formula is C 12 H 18 O5, chemical name is tough leather alkyd A, structural formula is as follows (1):

2. The compound according to claim 1, characterized in that The stereo configuration of the compound is shown in formula (2):

3. A method for extracting the compound according to claim 1 or 2, comprising the following steps: (1) inoculating the activated Lederma lucidum strain and Lederma fusca strain into a potato-glucose liquid medium, co-culturing and fermenting for a certain period of time under dark conditions, filtering and separating mycelium and fermentation liquid, soaking the mycelium with a solvent, mixing the soaking liquid with the fermentation liquid, and concentrating under reduced pressure to obtain a crude extract; (2) the crude extract is dissolved in water, extracted with ethyl acetate, and concentrated under reduced pressure to remove ethyl acetate to obtain an extract; (3) The extract obtained in step (2) was separated by normal phase silica gel column chromatography, using petroleum ether and acetone as eluents, eluting in a gradient of 20:0, 20:1, 1:1, and 0:1, v / v, and collecting the 1:1 elution fraction; (4) The eluted fractions collected in step (3) were separated by medium pressure liquid chromatography using reverse phase silica gel, with methanol and water as the eluent, eluting in a gradient ratio of 20:80, 40:60, 60:40, 80:20, and 100:0, v / v, and the eluate with a volume ratio of methanol to water of 40:60 was collected to obtain component B; (5) Component B was eluted by Sephadex LH-20 gel column chromatography, the eluent was pure acetone, and TLC was used for detection. The fluorescent spot under 254nm ultraviolet light was used as a reference, and vanillin-concentrated sulfuric acid-ethanol solution was used for color development. The fractions were combined according to the color spots to obtain two components, B1 and B2, of which the main spot of B2 was purple-red, R f The value is about 0.5 component, and the B2 component is passed through a normal phase chromatography column. The eluent is petroleum ether and acetone. The elution is carried out in a gradient of 6:1, 4:1, 2:1, 1:1, 0:1, v / v, and the components eluted at a volume ratio of petroleum ether to acetone of 4:1 are collected. After washing with petroleum ether, tough leather alcohol acid A is obtained.

4. The extraction method according to claim 3, characterized in that The potato-glucose liquid culture medium is a modified potato liquid culture medium, which contains, per liter, a boiled liquid prepared from 200 g of peeled potatoes, 20 g of glucose, 1 g of peptone, 1.5 g of KH2PO4, 1.5 g of MgSO4, and 10 mg of vitamin B1.

5. The extraction method according to claim 4, characterized in that The Leptospirillum pubescens strain and the Leptospirillum chinensis strain are both activated in a modified potato-glucose-agar medium, wherein the modified potato-glucose-agar medium is prepared by adding 17 g of agar per liter of modified potato liquid culture medium.

6. The extraction method according to claim 5, characterized in that In the step (1), equal amounts of the two activated strains are inoculated into a potato-glucose liquid culture medium, and cultured at a constant temperature of 25° C. in a dark environment for 25 days to complete fermentation; the solvent is a mixed solvent of chloroform and methanol in a volume ratio of 1:1, and the immersion extraction is performed 5 times, each time for 24 hours.

7. Use of the compound oleic acid A or a pharmaceutically acceptable preparation thereof according to claim 1 or 2 in the preparation of a drug for treating neurodegenerative diseases and / or a drug for treating pain.

8. The use according to claim 7, characterized in that: The drug for treating neurodegenerative diseases is a drug for treating Alzheimer's disease, Parkinson's disease, Huntington's disease and / or amyotrophic lateral sclerosis.

9. The use according to claim 7, characterized in that: The drug for treating pain is a drug for treating chronic migraine.

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