A compound obtained by co-culturing medicinal fungi Lederma lucidum and Lederma lucidum, and its preparation method and application

Abstract

This invention relates to the technical field of pharmaceutical compounds, specifically disclosing a compound isolated from the fermentation broth of a co-culture of the medicinal fungi Vibrahirsutin and Vibrahirsutin, its preparation method, and its medical uses. The compound is named vibrahirsutin A. The compound of the present invention exhibits significant neuroactivity and can be used to prepare new neuroprotective and analgesic drugs for the treatment of, but not limited to, cognitive impairment and bradykinesia associated with neurodegenerative diseases, as well as for pain relief. Results indicate that vibrahirsutin A significantly increases the frequency of spontaneous excitatory postsynaptic currents in neurons, with a half-maximal effective concentration of 23.96±0.016 mM. It also exhibits a significant analgesic effect in mice with a chronic migraine model induced by inflammatory decoction.

Description

Technical Field

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

[0002] Stereum hirsutum (Stereum hirsutum) is a wood-rotting fungus in the genus Stereum, belonging to the phylum Eumycota, subdivision Basidiomycotina, class Basidiomycetes, order Russulales, family Stereaceae. It is rich in terpene synthases, and over 40% of its secondary metabolites are sesquiterpenoids, which exhibit a variety of activities, including antibacterial, anti-inflammatory, antioxidant, anticancer, and cytotoxic activities. Other compounds include styrosin derivatives, sterols, aromatic acetylenes, and benzoates. In addition to the aforementioned activities, pancreatic lipase inhibition, α-glucosidase inhibition, glyoxalase inhibition, and phytotoxic activity have also been detected (Zhao Zhenzhu, Chemical composition and biological activities of six fungi, including matsutake, PhD dissertation, University of Chinese Academy of Sciences, 2018).

[0003] Boreosteum vibrans (Stereum genus) is a fungus of the same genus as Boreosteum. 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 compounds isolated from Boreosteum vibrans are vibralactone derivatives, with some sesquiterpenes and a few other compounds also found (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 tricholomacin derivatives and sesquiterpenes, but the yields are very different. C. tricholoma produces more sesquiterpenes, while C. fusca mainly produces tricholomacin 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 C. tricholoma and C. fusca after co-cultivation.

[0005] Neurodegenerative diseases adversely affect 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 behavioral, 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 physically, but also suffer greatly mentally, often accompanied by anxiety and depression, which seriously affects their 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, characterized by 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 migraines 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 lucidum, and to study the activity thereof.

[0009] The inventors of the present application systematically studied the chemical components of the co-culture fermentation products of the fungus Fischer-Lebensmittel and the fungus Fischer-Lebensmittel, and discovered the compound of formula (1), Fischer-Lebensmittel A. Studies on the neuropharmacological activity of Fischer-Lebensmittel A have shown that this molecule can significantly promote the generation of action potentials in pyramidal neurons of the insular cortex of mice, increase neuronal excitability, and enhance the frequency of spontaneous excitatory postsynaptic currents in the same type of neurons, suggesting that this compound has application prospects in the development of drugs for the treatment of neurodegenerative diseases. In addition, Fischer-Lebensmittel 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] In order to achieve the above-mentioned object of the present invention, the present invention provides a compound obtained by co-culturing the medicinal fungi Lederma lucidum and Lederma fuscae: 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) The activated Lederma lucidum strain and the Lederma lucidum strain are inoculated into a potato-glucose liquid medium at a ratio of 0.1 to 1:1, and after co-culturing and fermenting for a certain period of time under dark conditions, the mycelium and the fermentation liquid are filtered and separated, the mycelium is soaked in a solvent, the soaking liquid is mixed with the fermentation liquid, and then concentrated under reduced pressure to obtain a crude extract;

[0016] (2) The crude extract is dissolved in water, extracted with ethyl acetate, and concentrated 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 as the eluent, with methanol and water being eluted in a gradient of 20:80, 40:60, 60:40, 80:20, and 100:0, v / v, to obtain 12 fractions in total. 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 with pure acetone as the eluent and detected by TLC. The fluorescent spot under 254 nm ultraviolet light was used as a reference and the color was developed with vanillin-concentrated sulfuric acid-ethanol solution. The fractions were combined according to the color spots to obtain two components, B1 and B2. The main spot of component B2 was purple-red, and 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, and the elution is carried out in sequence according to the gradient of 6:1, 4:1, 2:1, 1:1, 0:1, v / v. The components eluted at a volume ratio of petroleum ether to acetone of 4:1 are collected, 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 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.

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

[0022] Furthermore, in step (1), equal amounts of two activated bacterial beads (each 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 drugs for treating neurodegenerative diseases and / or drugs 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 represented by formula (1), oleic acid A, is specifically manifested 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-maximal effective concentration (EC 50 ) value was 23.96±0.016 μM, and the neuronal sEPSC amplitude did not change before and after administration. Furthermore, the compound rennetic acid A can affect the excitability of pyramidal neurons in the mouse insular cortex, specifically promoting the frequency of neuronal action potential firing. Therefore, rennetic acid A has the potential to be used in the preparation of new drugs for treating neurodegenerative diseases.

[0027] The compound rennetic acid A also exhibits significant analgesic effects in mice modeling chronic migraine (CM) stimulated by inflammatory mediators. It significantly increases the acute mechanical withdrawal threshold and thermal withdrawal latency in the hind paw and periorbital regions of mice. Therefore, rennetic 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 Funding Project (No.CPT22033).

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

[0030] The present invention co-cultivates the medicinal fungi Lederma tricholoma and Lederma fusca, yielding a natural compound with a novel skeleton, a chemical structure completely different from any single-ingredient drug currently available on the market. Further development of this compound into a specialized treatment for neurodegenerative diseases and analgesia could circumvent patents on existing marketed drugs, providing a wider range of market options.

[0031] 2. The present invention reveals for the first time that the derivatives of the tenacin-type fungi (the biogenic precursor compound of tenacinol A is tenacin) 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 tenacin 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 NMR HSQC spectrum of the new compound obtained in Example 1 of the present invention;

[0038] Figure 6 This is the HMBC nuclear magnetic resonance spectrum of the new compound obtained in Example 1 of the present invention;

[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 This is the ROESY nuclear magnetic resonance spectrum of the new compound obtained in Example 1 of the present invention;

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

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

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

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

[0045] Figure 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] Unless otherwise specified, the following examples were performed under conventional conditions or those recommended by the manufacturer. All reagents and instruments used, unless the manufacturer is specified, were commercially available. Materials and reagents used were commercially available and analytically pure unless otherwise specified.

[0048] The animal experiments in the following examples have been approved by the Laboratory Animal and Biomedical Ethics Committee of South-Central University for Nationalities (No. 2019-SCUEC-AEC-021).

[0049] In the following examples: 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 deposited in the Ethnomedicine and Medicinal Fungi Research Group, School of Pharmacy, South-Central University for Nationalities, No. 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province. The public can purchase the strain from this unit.

[0050] Boreotereum vibrans was collected from the Kunming Botanical Garden in Yunnan Province and is the strain used in the paper "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). This strain is currently in the Ethnomedicine and Medicinal Fungi Research Group of South-Central University for Nationalities, under the registration number No. 20120920B. The address is: 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province. The strain is available for purchase from this institution.

[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 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, the same below) and sterilize them at 121°C for 25 minutes. Then inoculate S. hirsutum and B. vibrans (0.5 mm each) 2 The mycelium-containing agar blocks) were activated on potato-glucose-agar medium (prepared by adding 17 g of agar per liter of the above-mentioned modified potato liquid medium) plates, cultured at 25°C for 7 days, and when the mycelium grew to about 1 cm from the edge of the plate, 0.5 mm × 0.5 mm of mycelium-containing agar blocks were dug out and inoculated into the prepared modified 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 100 L 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 24 hours five times, filter after each soaking, combine the filtrates obtained five times, mix with the fermentation broth, and concentrate under reduced pressure to obtain a crude extract. Dissolve the crude extract in 2.5 L of water and extract with 5 L of ethyl acetate in a 10 L separatory funnel. After standing for 2 hours after shaking, collect the ethyl acetate layer, and continue to extract the lower aqueous layer with 5 L of ethyl acetate. Combine the collected ethyl acetate layers and concentrate using a rotary evaporator to obtain 36.33 g of extract.

[0054] Step 3, separating the obtained extract 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] The eluate obtained in step 4 and 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, methanol and water gradient elution, methanol to water volume ratio of 20:80, 40:60, 60:40, 80:20, 100:0, each gradient elution 1 L, collect the methanol to water volume ratio of 40:60 eluate 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 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. The fractions were combined according to the color spots to obtain two components, B1 and B2, of which 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-red, R f The value was approximately 0.5. Fraction B2 was further subjected to normal phase chromatography (petroleum ether-acetone, volume ratios of 6:1, 4:1, 2:1, 1:1, and 0:1) to obtain five subfractions. Fraction B2-2 was collected when eluted with a volume ratio of petroleum ether to acetone of 4:1. Thin-layer chromatography revealed a single purple-red spot. After merging and evaporation to remove the organic solvent, a colorless prismatic solid with a slightly yellow surface was obtained. The yellow substance on the surface was quickly removed by washing with petroleum ether to obtain crystalline compound 1 (47.5 mg), designated as oleic acid A.

[0057] Structural identification: Using modern spectroscopy techniques such as 1 H NMR spectroscopy, 13 The structure of the new compound obtained in step 5 was identified by C NMR, DEPT 135 NMR, two-dimensional 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) spectrum data (deuterated methanol)

[0060]

[0061] The 2D NMR, HMBC, and COSY spectra of the obtained compound are shown below:

[0062]

[0063] Then, the planar configuration of the obtained compound was determined by combining 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. Figure 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 assay was used to detect the cytotoxicity of compound 1 on neural cells N2a:

[0068] The CCK-8 assay was used to determine whether the test samples at concentrations 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 approximately 5 × 10 3Cells were seeded into 96-well plates in Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 10% fetal bovine serum and 1% penicillin / streptomycin, with a culture medium volume of 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 plates 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 (A group (an equal amount of DMSO was added and DMEM medium was supplemented to a final volume of 200 μL per well) was grown at 37°C and 5% CO for 24 hours. Then, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for another hour 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 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 no cells;

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

[0075] *Cell viability: cell proliferation activity or cytotoxic activity, in this example Figure 10 The cell viability is expressed in the middle.

[0076] The test results are as follows Figure 10 As shown, the compound of formula (1) showed no cytotoxicity to nerve cells N2a at a concentration of 1-100 μM, verifying the safety 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 hippocampal basket cell neurons in male rats.

[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-h dark / light cycle, and unlimited access to food and water. All animal experiments were conducted 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 potentials of pyramidal neurons in the insular cortex:

[0080] Experimental principle: The patch clamp technique uses a glass microelectrode to contact the cell membrane, connecting 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 patch clamp technique used in this example was performed as follows: All male mice were anesthetized with 2% isoflurane and rapidly decapitated. Preliminary trimmed brain tissue was immersed in an oxygen-saturated slicing solution (185 mM sucrose, 2.5 mM KCl, 2 mM MgSO₄, 2 mM CaCl₂, 26 mM NaHCO₃, 1.2 mM NaH₂PO₄, 25 mM glucose, pH 7.4) and ice-water. Coronal sections were cut using an oscillating microtome (Leica VT1200S) to a thickness of 300 μm. After dissection, brain slices were placed in an incubator containing oxygen-saturated ACSF (artificial cerebrospinal fluid): 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. The slices were incubated at 32-34°C for half an hour. The temperature was controlled by a TC-324C dual automatic temperature controller (Warner Instruments, Inc., USA) and continuously bubbled with 5% CO2 and 95% O2. Following incubation, the slices were transferred to 25°C for the experiment and data were recorded.

[0082] First, under an infrared differential interference microscope (Olympus, Japan), neurons with good activity were identified. Microelectrodes (3-8 MΩ) were then filled with electrode solution (122 mM potassium gluconate, 5 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 1 mM EDTA, 10 mM sodium phosphocreatine, 4 mM Na2ATP, 0.4 mM Na3GTP, pH 7.3). In bath mode, the microelectrode was manipulated using a micromanipulator to approach the neuron. When the resistance increased by 0.3-0.5 MΩ, the positive pressure was removed and an appropriate negative pressure was applied to allow the cell membrane to adhere to the opening of the electrode tip. When the seal resistance exceeded 1 GΩ, the negative pressure was removed, forming a high-resistance seal. Cell fast capacitance was compensated as needed, and appropriate negative pressure was continued until the cell membrane at the seal was broken. The microelectrode was then switched to patch mode to observe whether the resistance remained at or above 1 GΩ. Subsequent electrophysiological recordings were then performed in whole-cell mode.

[0083] Neurons were depolarized by stimulation with a 10pA gradient from 10 to 300pA for 1000ms, followed by stimulation with a 50-70pA current. Changes in action potential frequency were monitored and recorded before administration (final concentration 10μM, obtained by diluting the stock solution of the compound of formula (1) dissolved in DMSO and added to the electrode internal solution, with circulatory perfusion for 15 minutes), and after the drug was washed away with the electrode internal solution. Two-way analysis of variance was used to analyze the results. P < 0.05 was considered statistically significant.

[0084] Figure 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 sEPSCs in Pyramidal Neurons of the Mouse Insular Cortex:

[0086] Based on Experiment 2, 10 μM bicuculline (dissolved in DMSO) was added to the ACSF to block GABA receptors. The signal was filtered at 2 kHz and sampled at 20 kHz using an Axon 700B amplifier and an Axon 440A analog / digital interface (Mercury Molecular Instruments, Inc., USA). Neurons were depolarized by 10 pA gradient stimulation from 10 to 300 pA for 1000 ms. 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, and 100 μM drug concentration groups (all obtained by dissolving the compound of formula (1) in DMSO) and a control group (DMSO group). Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded before drug addition. Then, drug was added and the spontaneous excitatory post-synaptic currents were recorded again after 3-5 minutes. The experimental data were analyzed by one-way analysis of variance to analyze the differences between the groups. P < 0.05 was considered statistically significant.

[0087] The results are as follows Figure 12 As shown in Figure 3, 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 was determined by presynaptic factors, and its half-effective concentration (EC 50 ) value was 23.96±0.016μM (calculated based on the concentration and intensity of action, using Graphpad software, as shown in Figure 2). Figure 13 shown).

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

[0089] Chronic migraine (CM) often strikes middle-aged individuals, impacting their health and well-being, and imposing a heavy economic burden on both individuals and society. Medications for treating CM include analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), and triptans. In recent years, preventive medications, such as calcitonin gene-related peptide (CGRP), have been developed. However, treating CM is a long-term process, requiring both focused drug development and research into effective prevention or reversal of the structural and functional lesions caused by the disease.

[0090] In this example, an inflammatory soup model was established: a small incision was drilled into the back of the mouse's skull and an internal tube was inserted. After the mouse was released from anesthesia, an inflammatory soup (1 mM bradykinin, 1 mM 5-hydroxytryptamine agonist, 1 mM histamine, and a phosphate buffered saline solution containing 0.1 mM prostaglandin E2, the phosphate buffer used was 0.1M, pH = 7.4) was injected through the internal tube at a rate of 10 μL per dose, once every two days. After about 5 days (the third injection of inflammatory soup), the model was considered successful when the mouse's hind paw immediately withdrew when it touched the hot plate and the same side of the front paw immediately withdrew its head after scratching the face. The experimental group was administered Compound 1 (prepared with physiological saline to a stock solution of 1.4 μg / μL) to the inflammatory soup model mice once every two days, with each mouse receiving 14 μg. The experiment included a chemical drug positive control group (control, administered zolmitriptan tablets dissolved in saline at a concentration of 0.00405 g / kg, 10 μL per dose) and a model group (10 μL per dose of saline). Dosing was performed every two days. Following dosing, the therapeutic efficacy of the sample on chronic migraine was evaluated using the following experiments: Periorbital and hindpaw mechanical pain thresholds were measured using an electronic Von Frey mouse pain threshold meter (IITC Life Science, CA, USA). For periorbital mechanical pain threshold, the mice responded by avoiding the pain meter, shaking their heads, or pushing the meter away with their forepaws; for hindpaw mechanical pain threshold, the mice withdrew their paws or licked their paws. A plantar heat allergy meter (SA709, Jiangsu Saiangsi Biotechnology Co., Ltd.) applied a controlled temperature heat stimulus to the paws, and the latency to lift the paw was recorded. Measurements were taken seven times over 13 days.

[0091] The experimental results are shown in Figure 14 Compared with the model group, both the positive drug control group and the compound 1 administration group showed significant differences. Compound 1 significantly improved the acute mechanical withdrawal threshold of the mouse hind paw and periorbital area, and also significantly increased 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-cultivation of medicinal fungi Lederma tricholoma and Lederma fusca: chemical formula: 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) The activated Lederma lucidum strain and the Lederma lucidum strain were inoculated into a potato-glucose liquid medium, and after co-culturing and fermenting for a certain period of time under dark conditions, the mycelium and the fermentation liquid were filtered and separated, the mycelium was soaked in a solvent, the soaking liquid was mixed with the fermentation liquid, and then concentrated 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 the 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 reversed-phase silica gel, with methanol and water as the eluent, eluting in a gradient of 20:80, 40:60, 60:40, 80:20, and 100:0, v / v, and the eluate with a methanol to water volume ratio of 40:60 was collected to obtain component B; (5) Component B was eluted by Sephadex LH-20 gel column chromatography with pure acetone as the eluent and detected by TLC. The fluorescent spot under 254 nm ultraviolet light was used as a reference and the color was developed with vanillin-concentrated sulfuric acid-ethanol solution. The fractions were combined according to the color spots to obtain two components, B1 and B2. The main spot of component B2 was purple-red, and 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, and the elution is carried out in sequence according to the gradient of 6:1, 4:1, 2:1, 1:1, 0:1, v / v. The components eluted at the volume ratio of petroleum ether to acetone of 4:1 are collected, and after washing with petroleum ether, tough leather alcohol 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 strains of Ceratium pubescens and Ceratium chinense were both activated in a modified potato-glucose-agar medium, wherein the modified potato-glucose-agar medium was prepared by adding 17 g of agar per liter of modified potato liquid 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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