Preparation method and application of novel bird nest alkane diterpenoid compound

Abstract

The invention discloses a preparation method and application of a novel bird nest alkane diterpenoid compound, and relates to the technical field of medicinal chemistry. The invention aims to solve the problems that chemical synthesis needs harsh conditions and the cost is difficult to control when diterpenoid compounds are obtained in the prior art, and a large amount of resources are wasted and new molecules are difficult to obtain under conventional fermentation conditions. The prepared bird nest alkane type diterpene has dual functions of promoting neurite growth and nourishing neurons, is expected to be developed into a neuroprotective agent, and is used for treating neurodegenerative diseases and peripheral nerve injury diseases; meanwhile, the problems that in the prior art, when the compounds are obtained, conditions needed by chemical synthesis are harsh, the cost is difficult to control, a large number of resources are wasted under conventional fermentation conditions, but new molecules are difficult to obtain, and production and smooth follow-up biological activity research are not facilitated are effectively solved. The invention provides the preparation method and the application of the novel bird nest alkane diterpenoid compound.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a preparation method and application of a novel guanane diterpenoid compound. Background Art

[0002] Cyathane diterpenoids are compounds with a characteristic 5 / 6 / 7 three-membered ring hybrid skeleton, which are often found in liquid culture, solid culture and fruiting body of fungi. Most cyathane-xylosides are only found in liquid culture of fungi, while cyathane-non-xylosides can be extracted under a variety of culture conditions. Nearly 90% of cyathane diterpenoids have a highly conserved C-2-C-3 double bond (Tang HY, Yin X, Zhang CC, Jia Q, Gao JM. Structure diversity, synthesis, and biological activity of cyathane diterpenoids in higher fungi. Curr Med Chem. 2015, 22, 2375-91). This type of compound can promote neurite growth and neuronal nutrition, and has the biological effects of neurotrophic factors (Tang D, Xu YZ, Wang WW, Yang Z, Liu B, Stadler M, Liu LL, Gao JM. Cyathane Diterpenes from Cultures of the Bird's Nest Fungus Cyathus hookeriand Their Neurotrophic and Anti-neuroinflammatory Activities. JNat Prod. 2019, 28, 1599-1608). Its normal function of neurons and the nervous system is regulated by the levels of neurotrophic factors and their receptors. Bird's nest diterpenes have potential therapeutic value in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and peripheral nerve injury diseases such as spinal lateral sclerosis, cerebral infarction, and hypoxic-ischemic encephalopathy (QiJ, Gao YQ, Kang Sj, Liu C, Gao JM. Secondary metabolites of bird's nest fungi: chemical structures and biological activities. J Agr Food Chem. 2023, 71, 6513-6524). However, the chemical synthesis of bird's nest diterpenes has problems such as environmental pollution, reagent toxicity, and difficulty in removing chiral byproducts, resulting in high synthesis costs and environmental pollution.

[0003] In order to find new anti-neuroinflammatory drugs for the treatment of Alzheimer's disease, the study of cyclooxygenase-2 (COX-2) (Jiang Zhenzhou, Xu Dengqiu, Yang Hang, et al. Research progress on drug targets [J]. Pharmaceutical Progress, 2018, 42(11): 838-859) and nitric oxide synthase (NOS) (Shen Qin. 2006. Mechanism of inducible nitric oxide synthase gene transcription in inflammatory glial cells. Chinese Journal of Clinical Rehabilitation, 10(30): 101-104) activities has potential value. COX-2 is expressed in inflammatory sites and can cause neuronal apoptosis. It coexists with neurons containing neurofibrillary tangles (NFTs), indicating that increased expression of COX-2 in neurons may lead to neuronal death. Nitric oxide (NO) is an important bioactive substance that participates in the regulation of multiple physiological systems such as the cardiovascular system and the nervous system. Neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) (Nathan C, Xie QW. 1994. Regulation of biosynthesis of nitric oxide. J Biol Chem, 269: 13725-13728) and endothelial nitric oxide synthase (eNOS) are the three known nitric oxide synthase isozymes.

[0004] PC12 cells are cell lines cloned from rat adrenal pheochromocytoma and have general characteristics of neuroendocrine cells. They stop dividing under the induction of physiological levels of nerve growth factor (NGF), grow neurites, and differentiate into cells with sympathetic neuron characteristics. PC12 cells are widely used in the pathogenesis, neurophysiology, and neuropharmacology of Alzheimer's disease (Dai Jianying, Wang Hui, Liu Min, Hong Zhanying. Pathological models of Alzheimer's disease and progress in metabolomics research, Pharmaceutical Care and Research, 2021, 21(4), 241-320).

[0005] Chemical synthesis of guanane-containing diterpenes has problems such as environmental pollution, reagent toxicity, and difficulty in removing chiral byproducts. The high cost of synthesis and the environmental pollution caused are not conducive to the smooth progress of production. The separation of guanane-containing diterpenes under conventional fermentation conditions has a high repetition rate, resulting in unnecessary waste of resources. Summary of the invention

[0006] The purpose of the present invention is to solve the problems that in the past, chemical synthesis for obtaining diterpenoid compounds required harsh conditions, difficult to control costs, and a large amount of resources wasted under conventional fermentation conditions while it was difficult to obtain new molecules, and to provide a novel preparation method and application of guanane diterpenoid compounds.

[0007] A novel guanylate diterpenoid compound, wherein the novel guanylate diterpenoid compound comprises a novel guanylate diterpenoid compound 1 and a novel guanylate diterpenoid compound 2; the novel guanylate diterpenoid compound 1 has the structural formula:

[0008]

[0009] The structural formula of the novel guanidine diterpenoid compound 2 is:

[0010]

[0011] The preparation method of the novel guanane diterpenoid compound is carried out according to the following steps:

[0012] Step 1: inoculate the black egg nest fungus on a solid culture medium, the inoculation amount of the black egg nest fungus is 1-5%, and after culturing at 25-32° C. for 4-10 days, collect spores;

[0013] Step 2: inoculate the spores in step 1 into a primary liquid culture medium, and culture in a shake flask at 25-32° C. for 6-10 days to obtain a primary seed solution;

[0014] Step 3: inoculate the primary seed solution in step 2 into the secondary liquid culture medium at a ratio of 1:(10-50), and culture in a shake flask at 25-32° C. for 3-5 days to obtain the secondary seed solution;

[0015] Step 4: inoculate the secondary seed solution in step 3 into the fermentation medium at a ratio of 1:(10-50), culture in a shake flask at 25-32°C for 10-20 days, and collect the fermentation liquid;

[0016] Step 5: Extracting new guanosine diterpenoid compounds from the fermentation broth of step 4, wherein the new guanosine diterpenoid compounds include new guanosine diterpenoid compounds 1 and new guanosine diterpenoid compounds 2, and the mass ratio of the new guanosine diterpenoid compounds 1 to the new guanosine diterpenoid compounds 2 is 26:9.

[0017] The application of the above-mentioned novel guanylate diterpenoid compound, the application of the novel guanylate diterpenoid compound in the preparation of anti-neuroinflammatory drugs, the neuroinflammation includes neurodegenerative diseases and peripheral nerve injury diseases, the neurodegenerative diseases include but are not limited to Alzheimer's disease, Parkinson's disease and Huntington's disease, the peripheral nerve injury diseases include but are not limited to spinal lateral sclerosis, cerebral infarction and hypoxic-ischemic encephalopathy.

[0018] Beneficial effects of the present invention:

[0019] (1) The guanane-type diterpenes prepared by the present invention have the dual functions of promoting neurite growth and nourishing neurons, and are expected to be developed as neuroprotectants for the treatment of neurodegenerative diseases and peripheral nerve damage diseases; at the same time, they effectively solve the problems that the chemical synthesis required for obtaining such compounds in the past was relatively harsh, the cost was difficult to control, and a large amount of resources was wasted under conventional fermentation conditions while it was difficult to obtain new molecules, which were not conducive to the smooth progress of production and subsequent biological activity research.

[0020] (2) The preparation method of the present invention is simple to operate and has extremely high industrial production value, providing sufficient drug source supply for subsequent activity screening. In addition, the culture medium raw materials are easy to obtain and inexpensive, which is conducive to controlling production costs.

[0021] The invention can obtain a preparation method and application of a novel guanane diterpenoid compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 represents the 2D NMR correlation of the novel guanylate diterpenoid compound 1 and the novel guanylate diterpenoid compound 2, 1 represents the novel guanylate diterpenoid compound 1, and 2 represents the novel guanylate diterpenoid compound 2;

[0023] Figure 2 The H NMR spectrum of the novel guanane diterpenoid compound 1 is shown;

[0024] Figure 3 The NMR carbon spectrum of the novel guanane diterpenoid compound 1 is shown;

[0025] Figure 4 The HH COSY spectrum of the novel guanane diterpenoid compound 1 is shown;

[0026] Figure 5 The NMR HSQC spectrum of the novel guanane diterpenoid compound 1 is shown;

[0027] Figure 6 The HMBC NMR spectrum of the novel guanane diterpenoid compound 1 is shown;

[0028] Figure 7The NOESY spectrum of the novel guanane diterpenoid compound 1 is shown;

[0029] Figure 8 The H NMR spectrum of the novel guanane diterpenoid compound 2 is shown;

[0030] Fig. 9 The NMR carbon spectrum of the novel guanane diterpenoid compound 2 is shown;

[0031] Fig.10 The HH COSY spectrum of the novel guanane diterpenoid compound 2 is shown;

[0032] Fig.11 The NMR HSQC spectrum of the novel guanane diterpenoid compound 2 is shown;

[0033] Fig.12 The HMBC NMR spectrum of the novel guanane diterpenoid compound 2 is shown;

[0034] Fig.13 The NOESY spectrum of the novel guanane diterpenoid compound 2 is shown;

[0035] Fig.14 This is a graph showing the effects of two diterpenoid compounds of the present invention on LPS-induced NO production in BV-2;

[0036] Fig.15 This is a graph showing the effects of two diterpenoid compounds of the present invention on the protein levels of enzyme markers in BV-2 cells stimulated by LPS;

[0037] Fig.16 This is a graph showing the effect of two diterpenoid compounds of the present invention on promoting axon growth activity of PC-12 cells. DETAILED DESCRIPTION

[0038] Specific implementation method 1: This implementation method is a novel guanosine diterpenoid compound, wherein the novel guanosine diterpenoid compound is a novel guanosine diterpenoid compound 1 including a novel guanosine diterpenoid compound 2; the structural formula of the novel guanosine diterpenoid compound 1 is:

[0039]

[0040] The structural formula of the novel guanidine diterpenoid compound 2 is:

[0041]

[0042] Specific implementation method 2: This implementation method is a novel method for preparing guanane diterpenoid compounds, which is carried out according to the following steps:

[0043] Step 1: inoculate the black egg nest fungus on a solid culture medium, the inoculation amount of the black egg nest fungus is 1-5%, and after culturing at 25-32° C. for 4-10 days, collect spores;

[0044] Step 2: inoculate the spores in step 1 into a primary liquid culture medium, and culture in a shake flask at 25-32° C. for 6-10 days to obtain a primary seed solution;

[0045] Step 3: inoculate the primary seed solution in step 2 into the secondary liquid culture medium at a ratio of 1:(10-50), and culture in a shake flask at 25-32° C. for 3-5 days to obtain the secondary seed solution;

[0046] Step 4: inoculate the secondary seed solution in step 3 into the fermentation medium at a ratio of 1:(10-50), culture in a shake flask at 25-32°C for 10-20 days, and collect the fermentation liquid;

[0047] Step 5: Extracting new guanosine diterpenoid compounds from the fermentation broth of step 4, wherein the new guanosine diterpenoid compounds include new guanosine diterpenoid compounds 1 and new guanosine diterpenoid compounds 2, and the mass ratio of the new guanosine diterpenoid compounds 1 to the new guanosine diterpenoid compounds 2 is 26:9.

[0048] Specific implementation method three: The difference between this implementation method and specific implementation method two is that the black egg nest fungus described in step one is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The preservation date is August 2023 and the registration number is 5.2258.

[0049] The other steps are the same as those in the second specific implementation method.

[0050] Specific embodiment 4: The difference between this embodiment and specific embodiment 2 or 3 is that the solid culture medium described in step 1 contains 10% to 20% potato extract, 1% to 5% glucose and 1.5 to 2.5% agar.

[0051] The other steps are the same as those in the second or third embodiment.

[0052] Specific embodiment 5: The difference between this embodiment and specific embodiments 2 to 4 is that the primary liquid culture medium described in step 2 contains 10% to 20% potato extract and 1% to 5% glucose.

[0053] The other steps are the same as those in Specific Embodiments 2 to 4.

[0054] Specific embodiment 6: The difference between this embodiment and specific embodiments 2 to 5 is that the secondary seed liquid described in step 3 contains 10% to 20% potato extract, 1% to 5% glucose and 0.4 to 0.6% yeast extract.

[0055] The other steps are the same as those in Specific Embodiments 2 to 5.

[0056] Specific embodiment seven: The difference between this embodiment and any one of specific embodiments two to six is ​​that the fermentation medium described in step four contains 0.2% to 2% glucose, 0.2% to 2% molasses, 0.2% to 2% malt extract and 0.4 to 0.6% yeast extract.

[0057] The other steps are the same as those in Specific Embodiments 2 to 6.

[0058] Specific embodiment eight: This embodiment differs from specific embodiments two to seven in that: Step five extracts the novel guanidine diterpenoid compound from the fermentation broth in the following specific steps:

[0059] First, 0.05-0.1% volume fraction of formic acid is added to the fermentation liquid, and the bacterial cells are ultrasonically broken and then extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract; the ethyl acetate extract is concentrated under reduced pressure at 30-45°C to obtain an extract; the extract is redissolved with methanol, filtered to remove the residue, and then extracted with petroleum ether, and the petroleum ether layer is removed to obtain an extract; the extract is concentrated and then separated by column chromatography to obtain a novel guanane diterpenoid compound.

[0060] The other steps are the same as those in Specific Embodiments 2 to 7.

[0061] Specific embodiment 9: This embodiment differs from specific embodiments 2 to 8 in that the specific steps of column chromatography separation are as follows:

[0062] The concentrated extract is first separated by a normal silica gel column, and eluted with dichloromethane-methanol elution solvents with a volume ratio of dichloromethane to methanol of (100:1), (50:1), (20:1), (10:1), (5:1), (2:1), (1:1) and (0:1) to obtain a target fraction 1; the target fraction 1 is separated by an ODS column, and the elution solvent is acetonitrile with a gradient of 30% to 100%, and formic acid with a volume fraction of 0.05 to 0.1% is added to the organic phase to obtain a target fraction 2; the target fraction 2 is separated by a normal silica gel column, and petroleum ether and ethyl acetate are first used in a volume ratio of (10:1), (8:1 ), (6:1), (4:1), (2:1), (1:1) and (0:1) petroleum ether-ethyl acetate elution solvents are used for elution, and then dichloromethane-methanol elution solvents with a dichloromethane:methanol volume ratio of (100:1), (80:1), (60:1), (40:1), (20:1) and (10:1) are used for elution in sequence to obtain a target fraction 3; the target fraction 3 is finally separated by a preparative high performance liquid chromatography and a reverse phase chromatographic column to obtain a novel guanane diterpenoid compound, and the separation conditions are: 5-10 mL / min, 30%-60% acetonitrile, and the acetonitrile contains 0.1% by volume of formic acid.

[0063] The other steps are the same as those in Specific Embodiments 2 to 8.

[0064] Specific embodiment ten: This embodiment provides an application of a novel guanylate diterpenoid compound, and the application of the novel guanylate diterpenoid compound in the preparation of anti-neuroinflammatory drugs. The neuroinflammation includes neurodegenerative diseases and peripheral nerve injury diseases. The neurodegenerative diseases include but are not limited to Alzheimer's disease, Parkinson's disease and Huntington's disease. The peripheral nerve injury diseases include but are not limited to spinal lateral sclerosis, cerebral infarction and hypoxic-ischemic encephalopathy.

[0065] The following examples are used to verify the beneficial effects of the present invention:

[0066] Embodiment 1:

[0067] 1. The extraction method, identification, anti-neuroinflammatory assay and application of the compounds of this example are carried out according to the following steps:

[0068] 1. Experimental materials;

[0069] Reagents and instruments:

[0070] Commonly used organic solvents: chloroform, dichloromethane, methanol, ethyl acetate, petroleum ether and acetone are all industrial reagents. Organic solvents: DMSO, chromatographic methanol and chromatographic acetonitrile, etc.

[0071] Commonly used instruments: polarimeter Rudolph Autopol Ⅲ; high performance liquid chromatograph: LC-20AT; ultraviolet spectrometer: Thermo Evolution-300; infrared spectrometer: Bruker TENSOR 27 (potassium bromide tablet method); nuclear magnetic resonance: Bruker Avance Ⅲ500 (TMS internal standard); low resolution mass spectrometer: Thermo Fisher LTQ Fleet; high resolution mass spectrometer: Agilent 6520 Accurate-Mass Q-TOF LC / MS. Rotary evaporator: RE-2000A; low temperature coolant circulation pump: DLSB-10 / 20 (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.); circulating water multi-purpose vacuum pump: SHB-Ⅲ (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.); clean bench: SW-OJ-2F (Suzhou Antai Air Technology Co., Ltd., Sujing Group); vertical steam sterilizer: MLS-3780. Column chromatography silica gel (100-200 mesh, 200-300 mesh and 300-400 mesh) and thin layer chromatography silica gel (silica gel H) were all produced by Qingdao Ocean Chemical Plant; liquid chromatography column: Hypersil BDS 5μm C18 (250×4.6and 250×10; Thermo); hydroxypropyl dextran gel Sephadex LH-20 and RP-C18 reverse silica gel were all produced by Merck.

[0072] 2. Fermentation of Cyathus striatus:

[0073] Step 1: Inoculate a 9 mm diameter black egg nest fungus cake onto a solid culture medium, with the inoculation amount of black egg nest fungus being 5%. After culturing at 28°C for 7 days, collect spores with a cotton swab;

[0074] The strain number of the Cyathus striatus is CBPFE A06, which is deposited by Wei Jing of Shangluo College at the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is August 2023 and the registration number is 5.2258.

[0075] The solid culture medium contains 20% potato extract, 2% glucose and 1.5% agar.

[0076] Step 2: inoculate the spores in step 1 into a primary liquid culture medium, and culture in a shake flask at 28° C. for 7 days to obtain a primary seed solution;

[0077] The primary liquid culture medium contains 20% potato extract and 2% glucose.

[0078] Step 3: inoculate the primary seed solution in step 2 into the secondary liquid culture medium at a ratio of 1:20, and culture in a shake flask at 28°C for 3 days to obtain the secondary seed solution;

[0079] The secondary seed liquid contains 20% potato extract, 2% glucose and 0.5% yeast extract.

[0080] Step 4: inoculate the secondary seed solution in step 3 into the fermentation medium at a ratio of 1:40, culture in a shake flask at 28°C for 14 days, and collect the fermentation liquid;

[0081] The fermentation medium contains 0.5% glucose, 0.5% molasses, 0.4% malt extract and 0.4% yeast extract.

[0082] Extraction of new-structure guanane-type diterpenes from fermentation products of Trichoderma occidentalis:

[0083] Step 5: first add 0.05-0.1% volume fraction of formic acid to the fermentation broth, ultrasonically disrupt the bacteria, and then extract with an equal volume of ethyl acetate to obtain an ethyl acetate extract; the ethyl acetate extract is concentrated under reduced pressure at 30-45°C to obtain an extract; the extract is redissolved with methanol, filtered to remove the residue, and then extracted with petroleum ether, and the petroleum ether layer is removed to obtain an extract; after the extract is concentrated, it is loaded on a normal silica gel column (Sillica gel, 200-300 mesh) by mixing the sample, and the column is decompressed, and eluted with dichloromethane-methanol elution solvents with a volume ratio of dichloromethane to methanol of (100:1), (50:1), (20:1), (10:1), (5:1), (2:1), (1:1) and (0:1) in sequence to obtain 6 fractions A1-A6, and the fraction containing guanane-type diterpenes is detected by HPLC-MS.

[0084] The target fraction A5 was further loaded onto an ODS column, the elution solvent was 30% to 70% acetonitrile in gradient, and 0.05 to 0.1% by volume of formic acid was added to the organic phase, the retention time was 60 min, and 600 mg of a crude fraction A5C6 containing novel guanane diterpenes was obtained.

[0085] The crude fraction A5C6 was then separated by a normal phase silica gel column, firstly eluted with a petroleum ether-ethyl acetate elution solvent having a volume ratio of petroleum ether to ethyl acetate of (10:1), (8:1), (6:1), (4:1), (2:1), (1:1) and (0:1), and then eluted with a dichloromethane-methanol elution solvent having a volume ratio of dichloromethane to methanol of (100:1), (80:1), (60:1), (40:1), (20:1) and (10:1), and then detected by HPLC-MS, and the target fraction was taken and chromatographed by preparative high performance liquid chromatography (HPLC) and a reverse phase column (YMC-Park C18, 20×250mm, 5μm) for final separation; the separation conditions are: 5-10mL / min, 30%-60% acetonitrile, and the acetonitrile contains 0.1% formic acid by volume; the new structure of the new guanosine diterpenoid compound 1 and the new guanosine diterpenoid compound 2 are finally obtained in the form of yellow oil, with the weights of 26mg and 9mg respectively.

[0086] 2. Structural analysis of novel guanosine diterpenoids 1 and 2:

[0087] 6 mg of the novel guanane diterpenoid compound 1 was dissolved in 0.5 mL of methanol-d4 and 5 mg of the novel guanane diterpenoid compound 2 was dissolved in 0.5 mL of DMSO-d6, and the NMR data were collected using an Aglient DD2 600 MHz NMR nuclear magnetic resonance instrument. 1 H- and 13 The structure of the compound was determined by C-NMR data analysis, and the molecular formula of the compound was determined by high resolution mass spectrometry (HR-ESI-MS).

[0088] The structural formula of the novel guanane diterpenoid compound 1 is:

[0089]

[0090] The structural formula of the novel guanane diterpenoid compound 2 is:

[0091]

[0092] Figure 1 Shows the 2D NMR correlation of novel guanane diterpenoid compound 1 and novel guanane diterpenoid compound 2.

[0093] Figure 2 The NMR spectrum of the novel guanane diterpenoid compound 1 is shown. Figure 3 The NMR carbon spectrum of the novel guanane diterpenoid compound 1 is shown. Figure 4 The HH COSY spectrum of the novel guanane diterpenoid compound 1 is shown. Figure 5The NMR HSQC spectrum of the novel guanane diterpenoid compound 1 is shown. Figure 6 The HMBC NMR spectrum of the novel guanane diterpenoid compound 1 is shown. Figure 7 Shows the NMR NOESY spectrum of the novel guanane diterpenoid compound 1.

[0094] Figure 8 The NMR spectrum of the novel guanane diterpenoid compound 2 is shown. Fig. 9 The NMR carbon spectrum of the new guanane diterpenoid compound 2 is shown. Fig.10 The HH COSY spectrum of the novel guanane diterpenoid compound 2 is shown. Fig.11 The NMR HSQC spectrum of the novel guanane diterpenoid compound 2 is shown. Fig.12 The HMBC NMR spectrum of the novel guanane diterpenoid compound 2 is shown. Fig.13 Shows the NMR NOESY spectrum of the novel guanane diterpenoid compound 2.

[0095] Table 1 shows the novel guanane diterpenoid compound 1 1 H and 13 C and 2D NMR data;

[0096] Table 1

[0097]

[0098]

[0099] Table 2 shows the novel guanidine diterpenoid compound 2 1 H and 13 C and 2D NMR data;

[0100] Table 2

[0101]

[0102]

[0103] 3. Determination of the anti-neuritis activity of novel guanylate diterpenoids 1 and 2:

[0104] 1. Nitric oxide (Lactate dehydrogenase, NO) inhibitory activity:

[0105] NO is an important inflammatory mediator. In neurons and glial cells of the brain with neurodegenerative diseases, excessive production of NO is common, indicating that NO is involved in such neuroinflammation (FJ Jimenez-Jimenez, H. Alonso-Navarro, MT Herrero, E. Garcia-Martin, JA Agundez, 2016. An Update on the Role of Nitric Oxide in the Neurodegenerative Processes of Parkinson's Disease, Curr. Med. Chem. 23 (24): 2666-2679). The inhibitory effect of the compound on LPS-induced NO production in BV-2 microglia was evaluated. BV-2 cells (2 × 10 5 / mL) were inoculated in 96-well plates and cultured at 37°C for 24 hours. The experimental groups were divided into: control group (DMSO), LPS treatment group (1μg / mL LPS) and compound treatment group (1μg / mL LPS + 50μM compound), and the cells were incubated for another 24 hours after drug treatment. The nitrite concentration in the culture medium was measured by a commercial analytical kit according to the protocol. The main principle is to determine the anti-neuritis activity of the compound by detecting the release of NO in the culture medium by the Griess method. The supernatant of BV-2 cells (50μL) was reacted with Griess reagent (50μL Griess regent I and 50μL Griess regent II). The absorbance was measured at 540nm on a Bio-Tek microplate reader, and the nitrite concentration was calculated by the nitrite standard curve using sodium nitrite as the standard.

[0106] Inhibition rate % = (A LPS组 -A 实验组 ) / (A LPS组 -A 空白组 )×100;

[0107] Fig.14 Graph showing the effect of two diterpenoid compounds of the present invention on LPS-induced NO production in BV-2. Fig.14 As shown, after 24 hours of LPS stimulation of BV-2 cells, the production of NO increased by about 135%. Both novel guanane diterpenoids 1 and 2 had a strong inhibitory effect on the production of NO, among which novel guanane diterpenoid 2 had the strongest inhibitory activity, reducing the production of NO by 33.3%.

[0108] 2. Western blot analysis of the inhibitory effects of novel guanane diterpenoids 1 and 2 on the expression of iNOS and COX-2:

[0109] After drug treatment, cell proteins were extracted according to the method described in the literature (Cheng Y, Yang C, Zhao J, Tse HF, Rong J. Proteomic identification of calcium-binding chaperone calreticulin as a potential mediator for the neuroprotective and neuritogenic activities of fruit-derivedglycoside amygdalin. J Nutr Biochem. 2015, 26 (2): 146-154), and protein expression was analyzed by western blotting. In brief, protein samples were denatured by boiling with sample buffer, followed by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked in TBST solution containing 5% BSA at 4°C overnight, then incubated in a solution containing primary antibodies against iNOS and COX-2 (Cell signaling Technology, Boston, MA, USA) at 4°C for 4 h, rinsed three times with TBST, and then incubated in a solution containing secondary antibodies labeled with horseradish peroxidase at 4°C for 3 h. After washing the membrane, the test was performed according to the instructions of the ECL kit from GE Healthcare.

[0110] Fig.15 This is a graph showing the effect of the novel guanane diterpenoid compound 2 of the present invention on the level of enzyme marker protein in BV-2 cells stimulated by LPS. Fig.15 As shown in the figure, LPS significantly enhanced the expression of iNOS and COX-2. The novel guanosine diterpenoids 1 and 2 had a more obvious inhibitory effect on the expression of iNOS and COX-2, and the inhibitory effect of the novel guanosine diterpenoid 2 was more significant.

[0111] 3. NGF-dependent activity of the compound in promoting neurite outgrowth in PC-12 cells:

[0112] PC-12 single cell suspension was cultured at 2×10 4The cells were inoculated at a density of 100 μg / well in a poly-lysine-coated 24-well plate. After culturing for 24 hours until the cells adhered to the wall, the PC-12 cells were starved with low serum medium (containing 1% heat-inactivated horse serum and 0.5% heat-inactivated fetal bovine serum) and replaced with fresh low serum medium. The NGF stock solution was diluted with PBS (final concentration was 20 ng / ml), and the stock solution of the test compound was diluted with serum-free medium to different concentrations (10-40 μM), and added to the 24-well plate respectively. After 48 hours of drug treatment, the morphological changes of the cells were observed under an inverted microscope. Cells containing one or more axons and at least one axon length reaching 1 times the diameter of the cell body were considered positive cells, and the cells were counted in multiple random fields of view, each of which contained at least 100 cells. Each treatment was repeated 3 times in the experiment. The data were expressed as mean ± SD. SPSS software was used for one-way analysis of variance. Duncan's test was used for multiple comparisons. Data with P < 0.05 were considered significant (Zhang, CC et al. Chemical constituents from the mushroom Hericium erinaceus and their ability to stimulate NGF-mediated neurite outgrowth on PC12 cells. Bioorg. Med. Chem. Lett. 2015, 25, 5078-5082).

[0113] The formula for calculating cell differentiation rate is: cell differentiation rate = effective cell number / total cell number.

[0114] Fig.16 This is a graph showing the effect of two novel guanane diterpenoid compounds 1 and 2 on the axon growth of PC-12 cells. Fig.16 As shown, the novel guanane diterpenoid compounds 1 and 2 both showed the activity of promoting the growth of PC-12 neuronal processes at 5 μM. Compared with NGF, diterpenoid compounds 1 and 2 significantly increased the percentage of cells bearing neurites. Specifically, the promotion rates of compounds 1 and 2 on cell processes were 195.8% and 148.4, respectively.

[0115] In summary, the compound in this example can be developed as a potential lead compound and has the potential to be used in the treatment of neuroinflammatory diseases as a neuroinflammatory inhibitor.

Claims

1. A novel guanane diterpenoid compound, characterized in that The novel guanylate diterpenoid compounds include novel guanylate diterpenoid compound 1 and novel guanylate diterpenoid compound 2; the structural formula of the novel guanylate diterpenoid compound 1 is: The structural formula of the novel guanidine diterpenoid compound 2 is:

2. The method for preparing a novel guanane diterpenoid compound according to claim 1, characterized in that The preparation method is carried out according to the following steps: Step 1: inoculate the black egg nest fungus on a solid culture medium, the inoculation amount of the black egg nest fungus is 1-5%, and after culturing at 25-32° C. for 4-10 days, collect spores; Step 2: inoculate the spores in step 1 into a primary liquid culture medium, and culture in a shake flask at 25-32° C. for 6-10 days to obtain a primary seed solution; Step 3: inoculate the primary seed solution in step 2 into the secondary liquid culture medium at a ratio of 1:(10-50), and culture in a shake flask at 25-32° C. for 3-5 days to obtain the secondary seed solution; Step 4: inoculate the secondary seed solution in step 3 into the fermentation medium at a ratio of 1:(10-50), culture in a shake flask at 25-32°C for 10-20 days, and collect the fermentation liquid; Step 5: Extracting new guanosine diterpenoid compounds from the fermentation broth of step 4, wherein the new guanosine diterpenoid compounds include new guanosine diterpenoid compounds 1 and new guanosine diterpenoid compounds 2, and the mass ratio of the new guanosine diterpenoid compounds 1 to the new guanosine diterpenoid compounds 2 is 26:

9.

3. The method for preparing a novel guanane diterpenoid compound according to claim 2, characterized in that The black egg nest fungus described in step 1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is August 2023, and the registration number is 5.2258.

4. The method for preparing a novel guanane diterpenoid compound according to claim 2, characterized in that The solid culture medium described in step 1 contains 10% to 20% potato extract, 1% to 5% glucose and 1.5 to 2.5% agar.

5. The method for preparing a novel guanane diterpenoid compound according to claim 2, characterized in that The primary liquid culture medium described in step 2 contains 10% to 20% potato extract and 1% to 5% glucose.

6. The method for preparing a novel guanane diterpenoid compound according to claim 2, characterized in that The secondary seed solution described in step three contains 10% to 20% potato extract, 1% to 5% glucose and 0.4 to 0.6% yeast extract.

7. The method for preparing a novel guanane diterpenoid compound according to claim 2, characterized in that The fermentation medium described in step 4 contains 0.2% to 2% glucose, 0.2% to 2% molasses, 0.2% to 2% malt extract and 0.4% to 0.6% yeast extract.

8. The method for preparing a novel guanane diterpenoid compound according to claim 2, characterized in that Step 5 The specific steps of extracting the novel guanidine diterpenoid compound from the fermentation broth are as follows: First, 0.05-0.1% volume fraction of formic acid is added to the fermentation liquid, and the bacterial cells are ultrasonically broken and then extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract; the ethyl acetate extract is concentrated under reduced pressure at 30-45 DEG C to obtain an extract; the extract is redissolved with methanol, filtered to remove the residue, and then extracted with petroleum ether, and the petroleum ether layer is removed to obtain an extract; the extract is concentrated and then separated by column chromatography to obtain a novel guanane diterpenoid compound.

9. The method for preparing a novel guanane diterpenoid compound according to claim 8, characterized in that The specific steps of column chromatography separation are as follows: The concentrated extract is first separated by a normal silica gel column, and eluted with dichloromethane-methanol elution solvents with a volume ratio of dichloromethane to methanol of (100:1), (50:1), (20:1), (10:1), (5:1), (2:1), (1:1) and (0:1) to obtain a target fraction 1; the target fraction 1 is separated by an ODS column, and the elution solvent is acetonitrile with a gradient of 30% to 100%, and formic acid with a volume fraction of 0.05 to 0.1% is added to the organic phase to obtain a target fraction 2; the target fraction 2 is separated by a normal silica gel column, and petroleum ether and ethyl acetate are first used in a volume ratio of (10:1), (8:1 ), (6:1), (4:1), (2:1), (1:1) and (0:1) petroleum ether-ethyl acetate elution solvents are used for elution, and then dichloromethane-methanol elution solvents with a dichloromethane:methanol volume ratio of (100:1), (80:1), (60:1), (40:1), (20:1) and (10:1) are used for elution in sequence to obtain a target fraction 3; the target fraction 3 is finally separated by a preparative high performance liquid chromatography and a reverse phase chromatographic column to obtain a novel guanane diterpenoid compound, and the separation conditions are: 5-10 mL / min, 30%-60% acetonitrile, and the acetonitrile contains 0.1% by volume of formic acid.

10. The use of a novel guanane diterpenoid compound as claimed in claim 1, characterized in that The novel guanane diterpenoid compounds are used in the preparation of anti-neuroinflammatory drugs. The neuroinflammation includes neurodegenerative diseases and peripheral nerve injury diseases. The neurodegenerative diseases include but are not limited to Alzheimer's disease, Parkinson's disease and Huntington's disease. The peripheral nerve injury diseases include but are not limited to lateral sclerosis, cerebral infarction and hypoxic-ischemic encephalopathy.

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