Preparation method and application of diterpenoid compounds

Abstract

Preparation method and application of diterpenoid, relate to the application technical field of diterpenoid.The present application separates two new compounds of diterpenoid A and diterpenoid B from a strain of Hericium fermentation extract, and discloses chemical structural formula of the two compounds, and corresponding nuclear magnetic, mass spectrum, infrared and ultraviolet data, and its function of significantly inhibiting the production of inflammatory BV2 cell NO.Diterpenoid B has 24% of the promotion rate of axon growth of PC-12 cell, and simultaneously has significant inhibition of TLR4 / NF-κB signal path to inhibit downstream inflammatory proteins of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and diterpenoid A and diterpenoid B both show significant anti-neuroinflammatory activity.The present application can obtain the preparation method and application of diterpenoid.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of applications of diterpenoids, in particular to a preparation method and applications of diterpenoids. BACKGROUND

[0002] Alzheimer's disease (AD) is a primary central nervous system disease characterized by cognitive dysfunction and memory impairment, and patients mainly concentrate on the middle and old ages. Clinically, AD is mainly manifested as memory decline, language disorder, positioning disorder, emotional out-of-control and behavior disorder, and the pathological characteristics are mainly manifested as neuroinflammation in the brain, loss of neurons and synapses in specific brain regions. At present, the drugs for treating AD in the clinic mainly include central cholinesterase inhibitors such as rivastigmine, galantamine, donepezil, huperzine A and N-methyl-D-aspartate receptor antagonists such as memantine. However, as the AD condition aggravates, the neurons capable of releasing acetylcholine are less and less, so the current drugs cannot reverse the progression of AD, and the application in the clinic has certain limitations. So far, no revolutionary drug has appeared [1] .

[0003] COX has two isomers of COX-1 and COX-2, wherein COX-2 is mainly expressed in inflammatory cells and can cause inflammatory reactions and tissue damage. In the early stage of AD, COX-2 has been highly activated, and the COX-2 immune activity is increased in the pyramidal cells and hippocampus of the cerebral cortex, which indicates that COX-2 may be involved in the pathogenesis of Alzheimer's disease, and has been used as a biomarker for AD diagnosis and prevention and indicates the occurrence of AD. Based on the key role of COX-2 in the pathogenesis of AD, the specific COX-2 inhibitor is undoubtedly quite broad for preventing and treating AD [2] .

[0004] As an important messenger molecule, NO is involved in important neurophysiological activities such as learning, memory and immune defense. Under normal physiological conditions, the nervous system mainly realizes the regulation of the content of NO through the regulation of the activation and deactivation process of nNOS. Neuron injury, repeated hyperbaric oxygen exposure and striatal injury can cause overexpression of nNOS and produce excessive NO. Excessive NO in the nervous system is closely related to the occurrence and development of diseases such as cerebral ischemic injury, Alzheimer's disease and Parkinson's disease. As a signal element, NO induced by nNOS is also very important for the initiation of systemic inflammatory response, and inhibition of nNOS or knocking out the gene expressing nNOS can reduce the systemic inflammatory response and mortality of rats injected with heat shock protein (LPS). Inhibition of nNOS activity can regulate the content of NO in the nervous system, thereby producing a therapeutic effect on some nervous system diseases [3] .

[0005] Rat phcochromocytoma cell (PC12) is derived from rat adrenal medulla pheochromocytoma, and is an ideal cell for studying physiology, pathology and pharmacology of nerve cells. The cell has certain neuroendocrine properties, and can synthesize dopamine and norepinephrine and other catecholamine neurotransmitters, and store them in cell vesicles for release through exocytosis. At the same time, the cell membrane of PC12 cell has nerve growth factor (NGF) receptor, and can be transformed into neuron-like cells with neuron characteristics after being induced and stimulated by NGF. Because of the passable characteristics, PC12 cell is widely used in researches on AD pathogenesis, nerve physiology and nerve pharmacology [4] . SUMMARY

[0006] The application aims at solving the above technical problems, and provides preparation of diterpenoid compounds and application thereof.

[0007] The diterpenoid compound is diterpenoid compound A and diterpenoid compound B; the chemical formula of the diterpenoid compound A is C 20 H 32 O3, and the structural formula is:

[0008]

[0009] The chemical formula of the diterpenoid compound B is C 28 H 46 O8, and the structural formula is:

[0010]

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

[0012] Step one: inoculate Hericium erinaceus strain into a culture medium flat dish, activate and culture at a temperature of 25-28 DEG C for 7-10 days to obtain an activated fungus cake;

[0013] Step two: inoculate the activated fungus cake in step one into a fermentation bottle containing culture medium, and ferment at room temperature for 30 days to obtain a fermentation product, and the inoculation amount is 1%;

[0014] Step three: the fermentation product in step two is extracted 2-3 times using ethyl acetate to obtain a crude extract; the crude extract is separated by a silica gel column, and gradient elution is carried out using chloroform-methanol mixed solutions with a volume ratio of (100:1), (50:1), (25:1), (10:1) and (5:1) in sequence, and then elution is carried out using a methanol solution to obtain a fraction a containing the target product; the fraction a containing the target product is separated by a chromatographic column a to obtain a fraction b containing the target product; the fraction b containing the target product is further purified by a gel column a, a silica gel column a and a semi-preparative high performance liquid chromatograph a in sequence to obtain the diterpenoid compound A;

[0015] The fraction F4 is separated by a chromatographic column b to obtain a fraction c containing the target product; the fraction c containing the target product is sequentially subjected to a silica gel column b and a gel column b to obtain a fraction d containing the target product; finally, the fraction d containing the target product is subjected to a semi-preparative high performance liquid chromatograph b to obtain the diterpenoid compound B.

[0016] The application of the diterpenoid compound, and the application of the diterpenoid compound in the preparation of an anti-neuroinflammatory drug, an iNOS and COX-2 activity inhibiting drug, an NO production inhibiting drug of an inflammatory BV2 microglial cell, a TLR4 / NF-κB signal pathway inhibiting drug for inhibiting downstream inflammatory proteins and a PC-12 cell axon growth promoting activity drug.

[0017] The beneficial effects of the present application are as follows:

[0018] The present application separates two new compounds, a diterpenoid compound A and a diterpenoid compound B, from a Hericium erinaceus fermentation extract, discloses chemical structural formulas of the two compounds, and corresponding nuclear magnetic, mass spectrum, infrared and ultraviolet data, and the use of the two compounds for significantly inhibiting the NO production of an inflammatory BV2 cell. The diterpenoid compound B has a PC-12 cell axon growth promoting rate of 24%, and the diterpenoid compound B has a significant inhibiting effect on the TLR4 / NF-κB signal pathway for inhibiting downstream inflammatory proteins, iNOS and COX-2. The diterpenoid compound A and the diterpenoid compound B both have a significant anti-neuroinflammatory activity, and have an application prospect for treating neuroinflammatory diseases.

[0019] The present application provides a preparation method and application of a diterpenoid compound. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The hydrogen spectrum of the diterpenoid compound A in the present application is shown in the figure;

[0021] Figure 2 The carbon spectrum of the diterpenoid compound A in the present application is shown in the figure;

[0022] Figure 3 HRESIMS of diterpenoid compound A in the present application;

[0023] Figure 4 UV of diterpenoid compound A in the present application;

[0024] Figure 5 IR of diterpenoid compound A in the present application;

[0025] Figure 6 Hydrogen spectrum of diterpenoid compound B in the present application;

[0026] Figure 7 Carbon spectrum of diterpenoid compound B in the present application;

[0027] Figure 8 HRESIMS of diterpenoid compound B in the present application;

[0028] Figure 9 UV of diterpenoid compound B in the present application;

[0029] Figure 10 IR of diterpenoid compound B in the present application;

[0030] Figure 11 Effect of diterpenoid compound A and diterpenoid compound B in the present application on LPS-induced BV-2 producing NO;

[0031] Figure 12 Effect of diterpenoid compound A and diterpenoid compound B in the present application on LPS-stimulated BV-2 cell enzyme marker protein level;

[0032] Figure 13 Effect of diterpenoid compound A and diterpenoid compound B in the present application on promoting PC-12 cell axon growth activity. DETAILED DESCRIPTION

[0033] Embodiment 1: the diterpenoid compound in the present embodiment is diterpenoid compound A and diterpenoid compound B; the chemical formula of the diterpenoid compound A is C 20 H 32 O3, and the structural formula is:

[0034]

[0035] the chemical formula of the diterpenoid compound B is C 28 H 46 O8, and the structural formula is:

[0036]

[0037] Specific embodiment two: the preparation method of the terpenoid compound in the embodiment two is carried out according to the following steps:

[0038] Step one: the Hericium erinaceus strain is inoculated into a culture medium flat dish, and is activated and cultured at a temperature of 25-28 DEG C for 7-10 days to obtain a fungus cake after activation;

[0039] Step two: the fungus cake after activation in step one is inoculated into a fermentation bottle containing a culture medium, and is fermented at room temperature for 30 days to obtain a fermentation product, and the inoculation amount is 1%;

[0040] Step three: the fermentation product in step two is extracted with ethyl acetate for 2-3 times to obtain a crude extract; the crude extract is segmented by using a silica gel column, and is gradient eluted by using chloroform-methanol mixed solutions with a volume ratio of (100:1), (50:1), (25:1), (10:1) and (5:1) in turn, and then is eluted by using a methanol solution to obtain a fraction a containing a target product; the fraction a containing the target product is separated by using a chromatographic column a to obtain a fraction b containing the target product; and the fraction b containing the target product is further purified by using a gel column a, a silica gel column a and a semi-preparative high performance liquid chromatograph a in turn to obtain the terpenoid compound A.

[0041] The fraction F4 is separated by using a chromatographic column b to obtain a fraction c containing a target product; the fraction c containing the target product is sequentially subjected to a silica gel column b and a gel column b to obtain a fraction d containing a target product; and finally, the fraction d containing the target product is subjected to a semi-preparative high performance liquid chromatograph b to obtain the terpenoid compound B.

[0042] Specific embodiment three: the difference between the embodiment and the embodiment two is that the culture medium in step one is composed of 2% glucose, 0.2% yeast extract, 0.5% peptone, 0.05% MgSO4 and 0.1% KH2PO4.

[0043] The other steps are the same as those in the embodiment two.

[0044] Specific embodiment four: the difference between the embodiment and the embodiment two or three is that the culture medium in step two is composed of 100 g of rice and 60 mL of water.

[0045] The other steps are the same as those in the embodiment two or three.

[0046] Specific embodiment five: the difference between the embodiment and one of the embodiments two to four is that the solvent in the chromatographic column a in step three is a methanol-water mixed solution, and the mass fraction of methanol is 83%.

[0047] The other steps are the same as those in the embodiment two to four.

[0048] Specific embodiment six: the difference between this embodiment and one of specific embodiments two to five is that: the solvent in step three of gel column a is methanol; the solvent in silica gel column a is a mixed solution of petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is 6:4; the solvent in semi-preparative high performance liquid chromatograph a is a mixed solution of methanol and water, the mass fraction of methanol is 40%, and the flow rate of high performance liquid column is 2 mL / min.

[0049] The other steps are the same as specific embodiments two to five.

[0050] Specific embodiment seven: the difference between this embodiment and one of specific embodiments two to six is that: the solvent in step three of column b is a mixed solution of methanol and water, the mass fraction of methanol is 85%; the solvent in semi-preparative high performance liquid chromatograph b is a mixed solution of methanol and water, the mass fraction of methanol is 85%, and the flow rate of high performance liquid column is 2 mL / min.

[0051] The other steps are the same as specific embodiments two to six.

[0052] Specific embodiment eight: the difference between this embodiment and one of specific embodiments two to seven is that: the solvent in step three of silica gel column b is a mixed solution of chloroform and methanol, the volume ratio of chloroform to methanol is 25:1; the solvent in gel column b is methanol.

[0053] The other steps are the same as specific embodiments two to seven.

[0054] Specific embodiment nine: the application of the diterpenoid compound, the application of the diterpenoid compound in the preparation of anti-neuroinflammatory drugs, drugs for inhibiting the activity of nitric oxide synthase iNOS and cyclooxygenase COX-2, drugs for inhibiting the production of NO by inflammatory BV2 microglial cells, drugs for inhibiting the TLR4 / NF-κB signaling pathway to inhibit downstream inflammatory proteins, and drugs for promoting the activity of PC-12 cell axon growth.

[0055] Specific embodiment ten: the difference between this embodiment and specific embodiment nine is that: the neuroinflammation includes but is not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Parkinson's disease.

[0056] The other steps are the same as specific embodiment nine.

[0057] The following examples are used to verify the beneficial effects of the present application:

[0058] Example 1:

[0059] The preparation method of the diterpenoid compound is carried out according to the following steps:

[0060] Step one: inoculate Hericium erinaceus strain in culture medium flat dish, activate culture at 28℃ for 7 days to obtain activated fungus cake;

[0061] The Hericium erinaceus in step one is purchased from China General Microbiological Culture Collection Center (CGMCC) with the number 5.579, and the purchase website is https: / / cgmcc.net / directory / data?number=5.579&genus=&species=&yiming=&page=1;

[0062] The culture medium in step one is composed of 2% glucose, 0.2% yeast extract, 0.5% peptone, 0.05% MgSO4 and 0.1% KH2PO4.

[0063] Step two: inoculate the activated fungus cake (7mm 2 ) in step one into 500mL fermentation bottles containing culture medium, a total of 100 bottles, ferment at room temperature for 30 days to obtain fermentation product, inoculation amount is 1%;

[0064] The culture medium in step two is composed of 100g rice and 60mL water.

[0065] Step three: extract the fermentation product in step two with ethyl acetate for 3 times to obtain crude extract; use silica gel column to segment, and use chloroform-methanol mixed solution with volume ratio of (100:1), (50:1), (25:1), (10:1) and (5:1) of chloroform-methanol respectively for gradient elution, and then use methanol solution for elution to obtain six fractions F1, F2, F3, F4, F5 and F6; separate fraction F3 through chromatographic column a to obtain five fractions F3-1, F3-2, F3-3, F3-4 and F3-5; further purify fraction F3-3 through gel column a, silica gel column a and semi-preparative high performance liquid chromatograph a respectively to obtain diterpenoid compound A;

[0066] Note: The above fraction F3 and fraction F3-3 contain target product diterpenoid compound A, which is determined by nuclear magnetic resonance instrument and mass spectrometer.

[0067] Diterpenoid compound A is a new compound, named 16-carboxyl-13-epi-neoverrucosane, white powder, chemical formula is C 20 H 32 O3, and the structural formula is:

[0068]

[0069] Physical and chemical properties:

[0070] UV (MeOH): λmax (logε)=200(3.01)nm;

[0071] IR(KBr):ν max =3420,2924,1700,1530,1453,1384,1316cm -1 ;

[0072] HRESI-MS(positive)m / z:343.2240[M+Na] + ;

[0073] 1 H-NMR(500MHz,CD3OD)δ:4.00(1H,dd,J=10.8,7.3Hz,H-5),2.88(1H,m,H-15),2.35(1H,m,H-13),1.92(1H,m,H-12a),1.67(1H,m,H-12b),1.62(1H,m,H-6a),1.59(1H,m,H-14),1.50(1H,m,H-11a),1.41(1H,m,H-9a),1.32(1H,m,H-9b),1.29(1H,m,H-1),1.26(1H,d,J=7.0Hz,H-17),1.23(1H,m,H-8a),1.21(1H,m,H-11b),1.18(1H,s,H-18),1.07(1H,m,H-8b),0.96(2H,m,H-3),0.83(1H,s,H-20),0.80(1H,s,H-19),0.74(1H,m,H-6b),0.68(1H,m,H-2); 13 C-NMR(125MHz,CD3OD)δ:46.1(C-1),28.3(C-2),22.0(C-3),24.1(C-4),71.9(C-5),47.5(C-6),38.4(C-7),36.2(C-8),37.6(C-9),44.1(C-10),41.9(C-11),27.7(C-12),43.6(C-13),52.2(C-14),43.7(C-15),181.2(C-16),20.0(C-17),26.1(C-18),17,3(C-19),20.4(C-20)。

[0074] The fraction F4 was separated by the chromatographic column b to obtain five fractions F4-1, F4-2, F4-3, F4-4 and F4-5; the fraction F4-4 was sequentially passed through the silica gel column b and the gel column b to obtain three fractions F4-4-1, F4-4-2 and F4-4-3; finally, the fraction F4-4-1 was passed through the semi-preparative HPLC b to obtain the diterpenoid compound B.

[0075] Note: The above fraction F4, fraction F4-4 and fraction F4-4-1 contain the target product diterpenoid compound B, which is determined by a nuclear magnetic resonance instrument and a mass spectrometer.

[0076] The diterpenoid compound B is a new compound, named Erinacines L, white powder, chemical formula of C 28 H 46 O8, structural formula of:

[0077]

[0078] Physical and chemical properties:

[0079] UV (MeOH): λ max (logε) = 230 (2.90) nm;

[0080] IR (KBr): ν max = 3364, 2928, 2888, 1689, 1455, 1379, 1241 1194 cm -1 ;

[0081] HRESI-MS (positive) m / z: 533.3099 [M+Na] + ;

[0082] 1H-NMR (500 MHz, CD3OD) δ: 6.13 (1H, d, J = 8.0 Hz, H-13), 4.78 (1H, s, H-1'), 4.55 (1H, s, H-15), 4.26 (1H, d, J = 13.2 Hz, H-5'a), 3.88 (1H, d, J = 5.8 Hz, H-11), 3.82 (1H, m, H-3'), 3.80 (1H, m, H-2'), 3.75 (1H, m, H-4'), 3.48 (1H, m, H-14), 3.47 (1H, m, H-5'b), 3.36 (3H, s, H-22), 3.32 (3H, s, H-23), 3.29 (3H, s, H-21), 3.27 (1H, m, H-5), 2.98 (1H, m, H-18), 2.30 (2H, dt, J = 7.8, 1.4 Hz, H-2), 2.30 (1H, m, H-10a), 2.06 (1H, td, J = 12.9, 4.8 Hz, H-7b), 1.92 (1H, t, J = 13.6 Hz, H-10b), 1.52 (2H, t, J = 7.4 Hz, H-1), 1.45 (2H, m, H-8), 1.08 (1H, s, H-17), 1.00 (1H, td, J = 13.4, 4.2 Hz, H-7a), 0.97 (3H, d J = 4.6 Hz, H-20), 0.96 (3H, d J = 4.6 Hz, H-19), 0.78 (1H, s, H-16); 13 C-NMR (125 MHz, CD3OD) δ: 37.1 (C-1), 28.6 (C-2), 138.6 (C-3), 140.3 (C-4), 34.0 (C-5), 43.7 (C-6), 34.2 (C-7), 36.1 (C-8), 49.7 (C-9), 30.0 (C-10), 75.1 (C-11), 141.4 (C-12), 132.0 (C-13), 85.0 (C-14), 106.6 (C-15), 17.4 (C-16), 24.2 (C-17), 27.2 (C-18), 21.4 (C-19), 22.3 (C-20), 57.0 (C-21), 54.7 (C-22), 53.4 (C-23), 103.7 (C-1'), 68.5 (C-2'), 68.9 (C-3'), 70.0 (C-4'), 60.8 (C-5').

[0083] The solvent in the chromatographic column a is a mixture of methanol and water, and the mass fraction of methanol is 83%.

[0084] The solvent in the gel column a in step three was methanol; the solvent in the silica gel column a was a mixture of petroleum ether and ethyl acetate, with a volume ratio of 6:4; the solvent in the semi-preparative high performance liquid chromatograph a was a mixture of methanol and water, with a mass fraction of 40% methanol, and the flow rate of the high performance liquid column was 2 mL / min.

[0085] The solvent in the column b in step three was a mixture of methanol and water, with a mass fraction of 85% methanol; the solvent in the semi-preparative high performance liquid chromatograph b was a mixture of methanol and water, with a mass fraction of 85% methanol, and the flow rate of the high performance liquid column was 2 mL / min.

[0086] The solvent in the silica gel column b in step three was a mixture of chloroform and methanol, with a volume ratio of 25:1; the solvent in the gel column b was methanol.

[0087] Reagents and instruments:

[0088] Common organic solvents: chloroform, dichloromethane, methanol, ethyl acetate, petroleum ether and acetone, etc. are all industrial reagents. Organic solvents: DMSO, chromatographic methanol and chromatographic acetonitrile, etc.

[0089] Common instruments: polarimeter Rudolph Autopol III; high performance liquid chromatograph: LC-20AT; ultraviolet spectrometer: Thermo Evolution-300; infrared spectrometer: Bruker TENSOR 27 (potassium bromide tabletting method); nuclear magnetic resonance: Bruker Avance III 500 (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 cooling liquid circulating pump: DLSB-10 / 20 (Zhengzhou Great Wall Scientific Industrial and Trade Co., Ltd.); circulating water type multi-purpose vacuum pump: SHB-III (Zhengzhou Great Wall Scientific Industrial and Trade Co., Ltd.); clean bench: SW-OJ-2F (Suzhou Antai Air Technology Co., Ltd.); 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) are all produced by Qingdao Marine Chemical Plant; liquid chromatography column: Hypersil BDS 5 μm C18 (250×4.6 and 250×10; Thermo); hydroxypropyl dextran gel Sephadex LH-20 and RP-C18 reverse phase silica gel are both produced by Merk Company.

[0090] II. Experimental part:

[0091] 1. Nitric oxide (NO) inhibitory activity:

[0092] NO is an important inflammatory mediator. Overproduction of NO is common in neurons and glial cells of the brain with neurodegenerative diseases, suggesting that NO is involved in such neuroinflammatory reactions [5]. The inhibitory effect of compounds on LPS-induced NO production in BV-2 microglia was evaluated. BV-2 cells (2 × 10⁶ cells / year) were subjected to LPS-induced NO production before the addition of assay reagents. 5 Cells were seeded in 96-well plates at 37°C for 24 hours. The experimental groups consisted of a control group (DMSO), an LPS treatment group (1 μg / mL LPS), and a compound treatment group (1 μg / mL LPS + 50 μM compound). After treatment, cells were incubated for another 24 hours. Nitrite concentration in the culture medium was measured using a commercially available analytical kit according to the protocol. The main principle was to determine the anti-neuritis activity of the compound by detecting the amount of NO released from the culture medium using the Griess method. 50 μL of supernatant from BV-2 cells was reacted with Griess reagents (50 μL Griess reagent I and 50 μL Griess reagent II). Absorbance was measured at 540 nm using a Bio-Tek microplate reader, and nitrite concentration was calculated using a nitrite standard curve with sodium nitrite as the standard.

[0093] Inhibition rate % = (A LPS组 -A 实验组 ) / (A LPS组 -A 空白组 )×100;

[0094] Figure 11 This invention relates to the effects of diterpenoid compounds A and B on LPS-induced NO production from BV-2; for example... Figure 11 As shown, after LPS stimulation of BV-2 cells for 24 hours, both diterpenoid A and diterpenoid B exhibited strong inhibitory effects on NO production. Specifically, diterpenoid B inhibited the IC50 of NO production. 50 The value was 5.82 μM, which was much lower than the positive control quercetin of 15.88 μM.

[0095] 2. Western Blot Analysis:

[0096] After drug treatment, cell protein was extracted and analyzed by Western blotting according to the method described in reference [6]. Briefly, after denaturation by boiling in sample loading buffer, protein samples were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% BSA in TBST solution at 4°C overnight, then incubated in primary antibody (iNOS, COX-2 and GAPDH, Cell signaling Technology, Boston, MA, USA) solution at 4°C for 4 h, washed with TBST for 3 times, then incubated in secondary antibody (horseradish peroxidase-labeled) solution at 4°C for 3 h. After washing the membrane, the ECL kit from GE Healthcare was used to test according to the manufacturer's instructions.

[0097] Figure 12 The effects of diterpenoid compound A and diterpenoid compound B on the enzyme marker protein levels of BV-2 cells stimulated by LPS in the present application; as shown in Figure 12 LPS significantly enhanced the expression of iNOS and COX-2. Diterpenoid compound B had more obvious inhibitory effect on the expression of iNOS and COX-2, and the inhibition rates were 64.0% and 69.6%, respectively.

[0098] 3. The compound blocks the nuclear subunit p65 of NF-κB in BV-2 cells induced by LPS:

[0099] NF-κB is a well-characterized transcription factor and is considered to be a key factor in microglial cell-mediated neuroinflammation. Under normal circumstances, NF-kB is combined with inhibitory protein IκB as a p50 / p65 / IκB trimer and is restricted in the cytoplasm. Once activated by various stimulants such as LPS, IκB is phosphorylated by IκB kinase IKK, ubiquitinated, and then degraded. Therefore, the nuclear localization signal (NLS) on NF-κB is exposed, and then the molecule undergoes nuclear translocation. In the nucleus, NF-κB initiates the transcription of genes related to inflammatory factors [7] .

[0100] The nuclear translocation of NF-κB subunit p65 was detected as an indicator of NF-κB activation. Cells were collected for Western blotting. In order to detect the nuclear translocation of NF-κB p65, nuclear and cytoplasmic protein extraction kits were used to separate and extract nuclear and cytoplasmic fractions according to the instructions. The primary antibodies were: anti-NF-κB p65 (1:1000), anti-Lamin B1 (1:1000) and anti-GAPDH (1:1000).

[0101] As Figure 12As shown, LPS induction stimulation significantly increased the protein expression of phosphorylated NF-κB p65 in the nucleus, and diterpenoid B significantly inhibited the protein expression of phosphorylated NF-κB p65 stimulated by LPS, and the inhibition rate was 70.0%.

[0102] 4. Compound NGF-dependent PC-12 cell neurite outgrowth promoting activity:

[0103] PC-12 single cell suspension was inoculated into a polylysine-coated 24-well plate at a density of 2×10 4 cells per well, and the PC-12 cells were cultured for 24 h to adhere to the wall, then starved with a 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 20 ng / ml), and the test compound stock solution was diluted with serum-free medium to different concentrations (10-40 μM), and then added to the 24-well plate. After 48 h 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, each random field containing at least 100 cells. Each treatment was repeated 3 times in the test, and the data were expressed as mean ± standard deviation, one-way ANOVA was used for analysis, Duncan's test was used for multiple comparisons, and data with P<0.05 were significantly different [8] .

[0104] Cell differentiation rate calculation formula: cell differentiation rate = effective cell number / total cell number.

[0105] Figure 13 The effects of diterpenoid A and diterpenoid B on promoting PC-12 cell neurite outgrowth activity in the present application; as Figure 13 shown, diterpenoid A and diterpenoid B at 5 μM both showed PC-12 neurite outgrowth promoting activity. Compared with NGF, diterpenoid A and diterpenoid B significantly increased the percentage of neurite-bearing cells. Specifically, the cell differentiation rates of diterpenoid A and diterpenoid B were 13% and 24%, respectively.

[0106] In summary, the diterpenoid A and diterpenoid B prepared in the present application can be developed as potential lead compounds, and have potential for treating neuroinflammatory diseases as neuroinflammatory inhibitors.

[0107] References of the present application:

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Claims

1. Diterpene compounds characterized in that The diterpenoid compounds are diterpenoid compound A and diterpenoid compound B; the chemical formula of the diterpenoid compound A is C 20 H 32 O3, and the structural formula is: The chemical formula of the diterpenoid compound B is C 28 H 46 O8, and the structural formula is:

2. The method for preparing diterpenoid compounds according to claim 1, characterized in that... The preparation method is carried out according to the following steps: Step one: the Hericium erinaceus strain is inoculated in a culture medium flat dish, and is activated and cultured at a temperature of 25-28 DEG C for 7-10 days to obtain an activated fungus cake; Step two: the activated fungus cake in step one is inoculated in a fermentation bottle containing a culture medium, and is fermented at room temperature for 30 days to obtain a fermentation product, and the inoculation amount is 1%; Step three: the fermentation product in step two is extracted with ethyl acetate for 2-3 times to obtain a crude extract; the crude extract is subjected to gradient elution using chloroform-methanol mixed solutions with a volume ratio of chloroform to methanol of (100:1), (50:1), (25:1), (10:1) and (5:1) in sequence using a silica gel column, and then is eluted with a methanol solution to obtain a fraction a containing a target product; the fraction a containing the target product is separated through a chromatographic column a to obtain a fraction b containing the target product; the fraction b containing the target product is further purified through a gel column a, a silica gel column a and a semi-preparative high performance liquid chromatograph a in sequence to obtain the diterpenoid compound A; The fraction F4 is separated through a chromatographic column b to obtain a fraction c containing a target product; the fraction c containing the target product is subjected to a silica gel column b and a gel column b in sequence to obtain a fraction d containing a target product; Finally, the fraction d containing the target product is subjected to a semi-preparative high performance liquid chromatograph b to obtain the diterpenoid compound B.

3. The method of claim 2, wherein the method is a method of preparing a diterpene compound. The culture medium in step one is composed of 2% glucose, 0.2% yeast extract, 0.5% peptone, 0.05% MgSO4 and 0.1% KH2PO4.

4. The method of claim 2, wherein the method is a method of preparing a diterpene compound. The culture medium in step two is composed of 100 g of rice and 60 mL of water.

5. The method of claim 2, wherein the method is a method of preparing a diterpene compound. The solvent in the chromatographic column a in step three is a methanol-water mixed solution, and the mass fraction of methanol is 83%.

6. The method of claim 2, wherein the method is a method of preparing a diterpene compound. The solvent in the gel column a in step three is methanol; the solvent in the silica gel column a is a petroleum ether-ethyl acetate mixed solution, and the volume ratio of petroleum ether to ethyl acetate is 6:4; the solvent in the semi-preparative high performance liquid chromatograph a is a methanol-water mixed solution, the mass fraction of methanol is 40%, and the flow rate of the high performance liquid column is 2 mL / min.

7. The method of claim 2, wherein the method is a method of preparing a diterpene compound. The solvent in the chromatographic column b in step three is a methanol-water mixed solution, and the mass fraction of methanol is 85%; the solvent in the semi-preparative high performance liquid chromatograph b is a methanol-water mixed solution, the mass fraction of methanol is 85%, and the flow rate of the high performance liquid column is 2 mL / min.

8. The method of claim 2, wherein the method is a method of preparing a diterpene compound. The solvent in the silica gel column b in step three is a chloroform-methanol mixed solution, and the volume ratio of chloroform to methanol is 25:1; the solvent in the gel column b is methanol.

9. The application of the diterpenoid compound as described in claim 1, characterized in that... The diterpenoid compound is used for preparing an active drug for inhibiting nitric oxide synthase iNOS and cyclooxygenase COX-2, a drug for inhibiting the production of NO by inflammatory BV2 microglial cells, a drug for inhibiting the TLR4 / NF-κB signal pathway to inhibit downstream inflammatory proteins, and an active drug for promoting the growth of PC-12 cell axons.

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