CN117398370A - Preparation method and application of diterpenoid compound Libertellenone C - Google Patents
Preparation method and application of diterpenoid compound Libertellenone C Download PDFInfo
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- CN117398370A CN117398370A CN202311231269.6A CN202311231269A CN117398370A CN 117398370 A CN117398370 A CN 117398370A CN 202311231269 A CN202311231269 A CN 202311231269A CN 117398370 A CN117398370 A CN 117398370A
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- ethyl acetate
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Abstract
The invention relates to the technical field of medicines, and discloses a preparation method and application of diterpenoid compound Libertellenone C. The invention provides an application of diterpenoid compound Libertellenone C shown in formula (1) in preparing a medicine for protecting nerve cells in the anti-neurodegenerative disease. The diterpenoid compound Libertellenone C can inhibit activation of NLRP3 inflammatory corpuscles and regulate NF-kappa B P signal paths, plays a remarkable anti-neuritis role, and can be used for preparing medicaments for protecting nerve cells.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of diterpenoid compound Libertellenone C.
Background
Neurodegenerative diseases are chronic neurological dysfunctional disorders that gradually worsen with age, often due to loss of structure or function of neurons and or their myelin sheath. Two types are distinguished by their pathogenic phenotype, one is movement affecting, e.g., cerebellar ataxia; the other is Alzheimer's Disease (AD), etc., which affects memory and cognitive functions. The incidence of neurodegenerative diseases is increasing, AD and Parkinson's Disease (PD) are diseases occurring in front of the incidence of neurodegenerative diseases, mainly in middle-aged and elderly people, but the disease population of Huntington's Disease (HD) and amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS) has no concentrated age group, and chronic neurodegenerative diseases reach middle-late most irreversible conditions, so early discovery and early treatment are needed. Up to now, there is no better method for curing neurodegenerative diseases, which seriously affects the health and quality of life of human beings, and brings great economic burden to patients. The main clinical therapeutic drugs include brain cell activators such as piracetam, which can protect and repair brain nerve cells and enhance the brain cortex interconnecting function; nimodipine, verapamil, etc. as a calcium channel blocker capable of dilating cerebral vessels; antioxidants such as vitamin E, ginkgo preparations, melatonin, etc.; NMDA receptor antagonists memantine, cholinesterase inhibitors huperzine a, galanthamine, and the like; neurotrophic factor NTF, estrogen replacement therapy, and the like. However, these drugs have very limited therapeutic effects, but only delay the progression of the disease and cannot reverse the disease, so that development of safer and more effective low-cost small molecule drugs is needed.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a preparation method and application of diterpenoid Libertellenone C.
In order to achieve the above purpose, the first aspect of the present invention provides an application of diterpenoid compound Libertellenone C shown in formula (1) in preparing a medicament for protecting nerve cells in the anti-neurodegenerative disease.
The second aspect of the invention provides an application of diterpenoid compound Libertellenone C shown in formula (1) in preparing medicines for resisting neuroinflammation in neurodegenerative diseases.
Preferably, the preparation method of the diterpenoid compound Libertellenone C shown in the formula (1) comprises the following steps:
(1) Inoculating the Phaliota fungus on potato dextrose agar medium, and then culturing at constant temperature to obtain a seed culture medium;
(2) Cutting the seed culture medium into pieces, inoculating the pieces into a rice culture medium, and carrying out fermentation culture;
(3) Extracting the fermentation product obtained in the step (2) by ethanol, and concentrating under reduced pressure to obtain a total extract;
(4) Mixing the total extract with water, extracting with ethyl acetate, and concentrating under reduced pressure to obtain ethyl acetate extract;
(5) And separating the ethyl acetate extract by chromatography to obtain diterpenoid compound Libertellenone C shown in the formula (1).
Preferably, the Arthrospira fungus is Arthrospira rupestris.
Preferably, in step (1), the conditions of the constant temperature culture include: the temperature is 20-30deg.C, and the time is 5-12 days.
Preferably, in step (2), the conditions of the fermentation culture include: the temperature is 20-30deg.C, and the time is 30-40 days.
Preferably, in step (2), the solid-to-liquid ratio of rice and water in the rice culture medium is 180-220g:200ml.
Preferably, in step (3), the number of ethanol extractions is 8-15.
Preferably, in step (4), the number of ethyl acetate extractions is 8-13.
Preferably, the specific process of step (5) comprises:
(a) Subjecting the ethyl acetate extract to normal phase column chromatography, wherein the volume ratio is 20:1-0:1 in petroleum ether-ethyl acetate to obtain 5 components Fr.1-Fr.5;
(b) Subjecting the component Fr.5 to ODS column chromatography, and gradient eluting with methanol-water at volume ratio of 20:80-100:0 to obtain 5 components Fr.5.1-Fr.5.5;
(c) Subjecting the component Fr.5.2 to normal phase column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate with the volume ratio of 20:1-0:1 to obtain 5 components Fr.5.2.1-Fr.5.2.5;
(d) And separating and purifying the component Fr.5.2.2 by adopting high performance liquid chromatography to obtain the compound shown in the formula (1).
According to the invention, the ethyl acetate extract of the Arthrospira nucifera (Arthrinium arundinins) is separated and purified to obtain diterpenoid compound Libertellenone C (Libertellenone C), and through evaluation of anti-apoptosis activity, active oxygen generation inhibition activity, mitochondrial function protection activity and anti-neuroinflammation activity, the Libertellenone C can obviously resist the increase of intracellular active oxygen, the reduction of mitochondrial membrane potential and the reduction of apoptosis proportion caused by sodium glutamate, and the cell survival rate is improved. Meanwhile, libertillenone C has a remarkable anti-neuroinflammatory activity, and the mechanism of Libertillenone C probably relates to the inhibition of NO, iNOS, ROS, inflammatory factors and COX-2 production in microglial cells by NF- κB-P65/IκB channels.
Therefore, libertillenone C can inhibit the activation of NLRP3 inflammatory corpuscles and regulate NF- κBP65 signal path, exert remarkable anti-neuritis effect, and can be used for preparing medicaments for protecting nerve cells.
Drawings
FIG. 1 is a mass spectrum of Libertellenone C;
FIG. 2 is Libertellenone C 1 H NMR spectrum;
FIG. 3 is Libertellenone C 13 C NMR spectrum and DEPT spectrum
FIG. 4 is a graph showing the result of the protective effect of Libertillenone C on SH-SY5Y cells;
FIG. 5 is a graph showing the results of SH-SY5Y apoptosis in Libertillenone C in sodium glutamate injury model;
FIG. 6 is a graph showing the results of SH-SY5Y apoptosis in Libertillenone C in sodium glutamate injury model;
FIG. 7 is a graph showing the effect of Libertillenone C on the active oxygen content in SH-SY5Y cells;
FIG. 8 is a graph showing the effect of Libertillenone C on the mitochondrial membrane potential of SH-SY5Y cells;
FIG. 9 is a graph showing the results of toxicity detection of Libertillenone C on BV-2 cells and the effect of Libertillenone C on intracellular NO production;
FIG. 10 is a graph showing the effect of Libertillenone C on mRNA levels of BV-2 cells iNOS, HO-1, TNF- α, IL-1β, IL-6 and COX-2;
FIG. 11 is the result of Libertillenone C inhibiting activation of BV-2 intracellular NLRP3 inflammatory corpuscles.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the invention provides an application of diterpenoid compound Libertellenone C shown in formula (1) in preparing a medicine for protecting nerve cells in the anti-neurodegenerative disease.
The second aspect of the invention provides an application of diterpenoid compound Libertellenone C shown in formula (1) in preparing medicines for resisting neuroinflammation in neurodegenerative diseases.
The diterpenoid compound Libertellenone C is a white powdery compound obtained by separating and purifying ethyl acetate extract of the Philippine cinnabarina (Arthrinium arundinins) belonging to the genus Philippine, and the molecular formula is C 20 H 28 O 5 (obsd[M+Na] + at m/z 371.1829,caLibertellenone Cd[M+Na] + 371.1834)。
In a preferred aspect of the present invention, the preparation method of the diterpenoid compound Libertellenone C represented by formula (1) includes the following steps:
(1) Inoculating the Phaliota fungus on potato dextrose agar medium, and then culturing at constant temperature to obtain a seed culture medium;
(2) Cutting the seed culture medium into pieces, inoculating the pieces into a rice culture medium, and carrying out fermentation culture;
(3) Extracting the fermentation product obtained in the step (2) by ethanol, and concentrating under reduced pressure to obtain a total extract;
(4) Mixing the total extract with water, extracting with ethyl acetate, and concentrating under reduced pressure to obtain ethyl acetate extract;
(5) And separating the ethyl acetate extract by chromatography to obtain diterpenoid compound Libertellenone C shown in the formula (1).
Preferably, the Arthrospira fungus is Arthrospira rupestris.
The Rhizopus arundinaceus strain used in the invention is separated from centipede intestinal tracts, the sequence is amplified by an ITSrDNA method, blast comparison is carried out in a JGI database, the similarity with the sequence of the strain Arthrinium arundinins is 100%, thus the strain is determined to be Rhizopus arundinaceus, the strain is named Arthrinium arundinins TJ 414:414-1, and the strain is preserved in China center for type culture collection, with a preservation address: the preservation number of the Chinese Wuhan university is CCTCC NO: m20231516.
In step (1) of the present invention: inoculating the rhizoma anemones raddeanae strain to potato glucose agar medium (PDA culture medium) for constant temperature culture, and then subculturing the strain with good condition for 2-4 generations to obtain seed culture medium.
Preferably, in step (1), the conditions of the constant temperature culture include: the temperature is 20-30deg.C, and the time is 5-12 days.
In a specific embodiment, in step (1), the temperature of the constant temperature culture may be 20 ℃, 22 ℃,24 ℃, 26 ℃, 28 ℃, or 30 ℃, and the time of the constant temperature culture may be 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days.
In a specific embodiment, in step (1), the temperature of the constant temperature culture is 28 ℃, and the time of the constant temperature culture is 7 days.
Preferably, in step (2), the conditions of the fermentation culture include: the temperature is 20-30deg.C, and the time is 30-40 days.
In a specific embodiment, in step (2), the temperature of the fermentation culture may be 20 ℃, 22 ℃,24 ℃, 26 ℃, 28 ℃, or 30 ℃, and the time of the fermentation culture may be 30 days, 32 days, 34 days, 36 days, 38 days, or 40 days.
In a specific embodiment, in step (2), the temperature of the fermentation culture is 28 ℃, and the time of the fermentation culture is 30 days.
In a preferred aspect of the present invention, in the step (2), the solid-to-liquid ratio of rice and water in the rice culture medium is 180-220g:200ml, and the rice culture medium is further subjected to a sterilization treatment before inoculation, wherein the specific process of the sterilization treatment comprises: sterilizing rice culture medium at 120-130deg.C for 20-40min, and ultraviolet sterilizing for 15-30min.
In a specific embodiment of the present invention, the solid-to-liquid ratio of rice to water in the rice medium in step (2) may be 180g:200ml, 190g:200ml, 200g:200ml, 210g:200ml or 220g:200ml.
Further, in order to ensure aseptic operation, the operations of step (1) and step (2) in the present invention are performed on an ultra-clean bench.
In a preferred embodiment of the present invention, in step (3), the number of times of ethanol extraction is 8 to 15 times, and specifically, may be 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times.
In a preferred embodiment, in the step (3), during the ethanol extraction, the fermentation product is required to be completely soaked in ethanol for 1-2 days in each extraction process, and then the extract obtained in each step is decompressed and concentrated to obtain the total extract.
In a specific embodiment, the number of ethanol extractions in step (3) is 10.
In a preferred embodiment, the ethanol extraction in step (3) is performed with a concentration of ethanol of 90-98% by volume. Specifically, the concentration of ethanol may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% by volume.
In the step (4), when the total extract is mixed with water, the total extract is suspended in the water, then extracted by ethyl acetate, and then concentrated under reduced pressure to recover ethyl acetate, so as to obtain ethyl acetate extract.
Preferably, in step (4), the number of extraction with ethyl acetate is 8 to 13, and may be specifically 8, 9, 10, 11, 12 or 13.
In a specific embodiment, the number of ethyl acetate extractions in step (4) is 10.
Preferably, the specific process of step (5) comprises:
(a) Subjecting the ethyl acetate extract to normal phase column chromatography, wherein the volume ratio is 20:1-0:1 in petroleum ether-ethyl acetate to obtain 5 components Fr.1-Fr.5;
(b) Subjecting the component Fr.5 to ODS column chromatography, and gradient eluting with methanol-water at volume ratio of 20:80-100:0 to obtain 5 components Fr.5.1-Fr.5.5;
(c) Subjecting the component Fr.5.2 to normal phase column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate with the volume ratio of 20:1-0:1 to obtain 5 components Fr.5.2.1-Fr.5.2.5;
(d) And separating and purifying the component Fr.5.2.2 by adopting high performance liquid chromatography to obtain the compound shown in the formula (1).
In a specific embodiment of the present invention, in step (a), a petroleum ether-ethyl acetate mixed solvent with volume ratios of 20:1, 10:1, 5:1, 3:1, 1:1 and 0:1 is sequentially used as a mobile phase for gradient elution, and similar parts are detected and combined by TLC after gradient elution, so as to obtain 5 components fr.1, fr.2, fr.3, fr.4 and fr.5.
In a specific embodiment of the present invention, in the step (b), the eluent of the ODS column is a methanol-water mixed solution with a volume ratio of 20:80-100:0, and the similar fractions are combined by TLC detection after gradient elution, to obtain 5 components Fr.5.1, fr.5.2, fr.5.3, fr.5.4 and Fr.5.5.
In a specific embodiment of the present invention, in step (c), the eluent of the normal phase column chromatography is in a volume ratio of 20:1-0:1, and combining similar parts by TLC detection after gradient elution to obtain 5 components Fr.5.2.1, fr.5.2.2, fr.5.2.3, fr.5.2.4 and Fr.5.2.5.
In a preferred embodiment, in step (d), the mobile phase of the high performance liquid chromatography is a methanol-water mixed solvent with a volume ratio of 40:60 to 60:40.
The present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.
All data below are expressed as mean ± standard deviation (x±s) (at least three independent experiments). Histogram statistics were plotted using Graphpad Prism 8 software and differences between the different dosing groups were compared using one-way analysis of variance (ANOVA), P <0.05 being significant. The result obtained by the flow instrument is analyzed by BD Accuri C6 software; western Blotting results were used for cleavage of the bands using Image Studio Ver5.2, selection, adjustment of exposure, and quantitative analysis of the bands using Image J.
The room temperature described below is referred to as 25 ℃.
The Rhizopus arundinaceus strain used in the example is obtained by separating from centipede intestinal tracts, amplifying sequences by an ITSrDNA method, performing Blast comparison in a JGI database, and determining that the similarity with the sequence of the strain Arthrinium arundinins is 100%, thereby determining the Rhizopus arundinaceus strain as Arthrinium arundinins TJ 414:414-1, and preserving the strain in China center for type culture collection (CCTCC NO: m20231516.
Example 1
(1) Inoculating the recovered Rhizopus arundinacea strain TJ414-1 to a PDA culture medium, placing the PDA culture medium into a constant temperature cabinet at 28 ℃ for constant temperature culture for 7 days, and then carrying out subculture on the strain with good state, and culturing for 2-4 generations to obtain a seed culture medium with the strain;
(2) Adding 200g of rice and 200mL of distilled water into a 1L conical flask, sterilizing in an autoclave at 125 ℃ for 35min, then placing into an ultra-clean workbench for ultraviolet sterilization for 20min to obtain a rice solid culture medium, chopping the seed culture medium with the strain into the rice solid culture medium, uniformly mixing, sealing by a sealing film, and fermenting and culturing for 30 days at 28 ℃; the whole operation process of the steps (1) and (2) is carried out on an ultra-clean workbench;
(3) Extracting the fermentation product obtained in the step (2) with industrial ethanol with the concentration of 95 vol% for 10 times, completely soaking the fermentation product in ethanol in each extraction process for 1 day, and concentrating the extract obtained in each step under reduced pressure to obtain total extract;
(4) Mixing the total extract with water, suspending the total extract in the water when the total extract is mixed with the water, extracting for 10 times by adopting ethyl acetate, and concentrating under reduced pressure to recover ethyl acetate to obtain ethyl acetate extract;
(5) Subjecting the ethyl acetate extract to normal phase silica gel column chromatography, sequentially adopting petroleum ether-ethyl acetate mixed solvents with volume ratios of 20:1, 10:1, 5:1, 3:1, 1:1 and 0:1 as mobile phases for gradient elution, and detecting and combining similar parts by TLC after gradient elution to obtain 5 components Fr.1, fr.2, fr.3, fr.4 and Fr.5;
(6) Subjecting the component Fr.5 to ODS reversed phase column chromatography, gradient eluting with methanol-water mixed solution with volume ratio of 20:80-100:0, detecting and combining similar parts by TLC after gradient eluting to obtain 5 components Fr.5.1, fr.5.2, fr.5.3, fr.5.4 and Fr.5.5;
(7) Subjecting the component Fr.5.2 to normal phase silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate mixed solution with the volume ratio of 20:1-0:1, and detecting and combining similar parts by TLC after gradient elution to obtain 5 components Fr.5.2.1, fr.5.2.2, fr.5.2.3, fr.5.2.4 and Fr.5.2.5;
(8) And separating and purifying the component Fr.5.2.2 by adopting high performance liquid chromatography (the mobile phase is a methanol-water mixed solvent with the volume ratio of 40:60-60:40) to obtain the compound shown in the formula (1).
Detecting the obtained compound by nuclear magnetic resonance NMR and mass spectrometer as shown in figures 1-3, wherein figure 1 is mass spectrum of the obtained compound, and figure 2 is mass spectrum of the obtained compound 1 An H NMR spectrum, FIG. 3 shows the obtained compound 13 C NMR spectrum and DEPT spectrum, and data of obtained spectrumThe structure of the compound obtained in example 1 was determined by comparison with the nuclear magnetic resonance data of the known compound, and the result showed that the compound represented by formula (1) obtained in example 1 had a molecular formula of C 20 H 28 O 5 (obsd[M+Na]+at m/z 371.1829,caLibertellenone Cd[M+Na]+ 371.1834) is a diterpenoid compound Libertellenone C.
Test example 1 method for evaluating the neuroprotective Activity and studying the mechanism of action of the Compound obtained in example 1
CCK-8 assay to detect cell viability
1. SH-SY5Y: SH-SY5Y cells well grown were collected and diluted to a density of 1.5X10 5 The cell suspension of each mL was inoculated into a 96-well plate, after stable growth for 24 hours, a test compound diluted with DMEM/F12 medium (the compound represented by formula (1) obtained in example 1, libertellenone C, denoted as LC) was added so that the final concentration of the test compound in the well plate was 40, 20, 10, 5, 2.5. Mu.M, respectively, and a blank control group (i.e., no test compound was added) was set, incubated for 4 hours, a suitable amount of test solution was added according to the instructions of CCK-8 reagent, absorbance OD was measured after incubation with cells at 37℃for 1 hour, toxicity of the test compound was evaluated according to the survival rate (%) =OD test/OD control×100%, and the results of adding test compound of different concentrations are shown in A in FIG. 4.
2.1 collecting and diluting well-grown SH-SY5Y cells to a density of 1.5X10 5 The cell suspension of each mL was inoculated into a 96-well plate, and after stable growth for 24 hours, a test compound diluted with DMEM/F12 medium (the compound represented by formula (1) obtained in example 1, libertillenone C, denoted as LC) was added so that the final concentration of the test compound in the plate became 10 and 5. Mu.M, and after incubation for 4 hours, H diluted with DMEM/F12 medium was added 2 O 2 To make H in the pore plate 2 O 2 Final concentration of 500. Mu.M, and provided with a hydrogen peroxide model group (i.e., without the addition of test compound, directly with H diluted with DMEM/F12 medium 2 O 2 To make H in the pore plate 2 O 2 Final concentration of 500 μm), incubated together for 24 hours, then rootAdding a proper amount of test solution according to the instruction of the CCK-8 reagent, incubating the test solution and cells at 37 ℃ for 1 hour, and measuring absorbance OD;
2.2 collecting and diluting well-grown SH-SY5Y cells to a density of 1.5X10 5 Inoculating a cell suspension of a number/mL into a 96-well plate, adding a compound to be tested diluted with a DMEM/F12 medium (the compound shown as a formula (1) obtained in example 1, libertillenone C, which is marked as LC) after stable growth for 24 hours, so that the final concentration of the compound to be tested in the well plate is 10 and 5 mu M, adding sodium glutamate (Glu) diluted with the DMEM/F12 medium after incubation for 4 hours, so that the final concentration of sodium glutamate in the well plate is 20mM, and providing a sodium glutamate model group (i.e. without adding the compound to be tested, directly adding sodium glutamate diluted with the DMEM/F12 medium, so that the final concentration of sodium glutamate in the well plate is 20 mM), incubating for 24 hours, adding a proper amount of test solution according to the instructions of a CCK-8 reagent, incubating with the cells for 1 hour at 37 ℃, and measuring absorbance OD;
a blank control group is also arranged, namely, no compound to be detected, hydrogen peroxide and sodium glutamate are added;
test compounds were evaluated for H by cell viability (%) = (OD test-OD hydrogen peroxide model)/(OD blank-OD hydrogen peroxide model) ×100% 2 O 2 Protective effect after stimulation; the results of the blank, hydrogen peroxide model and test compounds at different concentrations after hydrogen peroxide stimulation are shown in figure 4, panel B;
the protective effect of the test compound after sodium glutamate stimulation is evaluated by the cell viability (%) = (OD test-OD sodium glutamate model)/(OD blank-OD sodium glutamate model) ×100%; the results of the blank, sodium glutamate model and different concentrations of the test compound after sodium glutamate stimulation are shown in graph B of fig. 4;
as shown in fig. 4, wherein, ### P＜0.0001， ### a blank group having P < 0.001vs. P < 0.0001, a sodium glutamate model group having P < 0.001vs. sodium glutamate model group, the compound of formula (1) obtained in example 1 being substantially non-cytotoxic to neuroblastoma cell SH-SY5Y, andthe method has a certain effect of improving the survival rate of the cell SH-SY5Y in two oxidative damage models, and has an outstanding effect in a sodium glutamate model, so that the sodium glutamate model is selected for subsequent experiments; wherein the compound of formula (1) obtained in example 1 can very significantly combat the decrease in SH-SY5Y cell viability caused by sodium glutamate at 10. Mu.M and 5. Mu.M.
Annexin V/PI double-staining method for detecting apoptosis
SH-SY5Y cells well grown were collected and diluted to a density of 1X 10 6 The cell suspension of each mL was inoculated into a 6-well cell culture plate, after the cell suspension was grown stably for 24 hours, the compound represented by formula (1) obtained in example 1 diluted with DMEM/F12 medium was added so that the final concentration of the compound represented by formula (1) in the well plate was 10, 5, 2.5. Mu.M, respectively, after 4 hours, an appropriate amount of sodium glutamate mother liquor (sodium glutamate diluted with DMEM/F12 medium) was added so that the final concentration of sodium glutamate in the well plate was 20mM, and a blank group (i.e., control group, without adding the compound represented by formula (1) and sodium glutamate) and a sodium glutamate model group (i.e., without adding the compound represented by formula (1)) were provided, sodium glutamate mother liquor is added only to make final concentration of sodium glutamate in the pore plate be 20 mM), after 24 hours, an Annexin V-FITC/PI apoptosis kit (KGA 108, keyGen Biotech Co, ltd.) is adopted to detect apoptosis, a blank group apoptosis is shown in a graph in FIG. 5, a sodium glutamate model group apoptosis is shown in a graph B in FIG. 5, apoptosis of a compound treatment shown in a formula (1) with concentration of 10, 5 and 2.5 mu M is shown in a graph C, D, E in FIG. 5, and the apoptosis rate of each group is calculated according to the ratio of apoptotic cells recorded in A1, B1, C1, D1 and E1 by using three parallel samples, and the result is shown in a graph F in FIG. 5;
according to fig. 5, there is, among other things, #### the proportion of early apoptotic cells in the sodium glutamate model group is obviously increased from 1.7% to 35.7% compared with normal cells, and after being treated by the compounds (10, 5 and 2.5 mu M) shown in the formula (1) with different concentrations, the early apoptotic cell number is respectively reduced to 11.3%, 18.1% and 25.3% at a certain dosageThe dependence demonstrates that the compound of formula (1) exerts a significant neuroprotective effect against 20mM sodium glutamate-induced SH-SY5Y apoptosis.
DAPI staining method for observing nuclear changes
SH-SY5Y cells well grown are collected and diluted to a density of 5X 10 5 Cell suspensions of each mL were inoculated into a 12-well plate, after 24 hours, the compound of formula (1) obtained in example 1 diluted with DMEM/F12 medium was added so that the final concentration of the compound of formula (1) in the well plate was 10, 5, 2.5. Mu.M, respectively, after 4 hours, an appropriate amount of sodium glutamate mother liquor (sodium glutamate diluted with DMEM/F12 medium) was added so that the final concentration of sodium glutamate in the well plate was 20mM, and a blank control group (i.e., the compound of formula (1) and sodium glutamate were not added) and a sodium glutamate model group (i.e., the compound of formula (1) was not added, and only sodium glutamate mother liquor was added so that the final concentration of sodium glutamate in the well plate was 20 mM) were added, and then DAPI staining solution (10mL,Beyotime Biotechnology) was applied so as to observe the morphology of SH-SY5Y nuclei, and the morphology of nuclei of the blank control group nuclei was shown in FIG. 6A, and the morphology of nuclei of the sodium glutamate model group was shown in FIG. 6B, and the morphology of nuclei of the compound of formula (1) was shown in FIG. 6 were treated in FIG. C, D, E; wherein white represents white light shooting, the morphology of cells can be seen, and DAPI can be seen as the morphology of cell nuclei
As shown in FIG. 6, the whole fluorescence intensity of the cells of the blank group after DAPI staining is weaker, the staining is even, the cell nucleus morphology is round and regular, the fluorescence intensity of the sodium glutamate model group is enhanced greatly, the cell nucleus is shrunken, the cell number is smaller, the cell damage caused by 20mM sodium glutamate solution is improved to different degrees by the three administration groups added with the compound shown in the formula (1), the whole fluorescence intensity is close to that of the blank group, and the cell morphology is normal. Meanwhile, 20mM sodium glutamate can be directly observed under a microscope to obviously cause the reduction of the total cell number and the adherent cell number of SH-SY5Y, the cell morphology is also greatly changed, the normal long shuttle shape is changed into a round shape, and the administration group treated by the compound shown in the formula (1) with 10, 5 and 2.5 mu M is given in advance, so that the cell damage caused by 20mM sodium glutamate can be obviously improved, the cell morphology is increased compared with the sodium glutamate model group, the cell morphology tends to the normal control group, and the protection effect is obvious at the concentration of 10 mu M and 5 mu M.
DCFH-DA staining method for detecting content of active oxygen in cells
SH-SY5Y cells well grown were collected and diluted to a density of 1X 10 6 The cell suspension of each mL was inoculated into a 6-well cell culture plate, after culturing for 24 hours, the compound of formula (1) obtained in example 1 diluted with DMEM/F12 medium was added so that the final concentration of the compound of formula (1) in the well plate was 10, 5, 2.5. Mu.M, respectively, after 4 hours, an appropriate amount of sodium glutamate mother liquor (sodium glutamate diluted with DMEM/F12 medium) was added so that the final concentration of sodium glutamate in the well plate was 20mM, and a Control group (i.e., control group, without adding the compound of formula (1) and sodium glutamate) and a sodium glutamate model group (i.e., without adding the compound of formula (1) so that the final concentration of sodium glutamate in the well plate was 20 mM) were added, after 24 hours, the cells were collected and tested according to the instructions of an active oxygen test kit (Solarbio Life Sciences), the results of the sodium glutamate model group were shown in FIG. 7A, the results of the sodium glutamate model group were shown in FIG. 7B, the compounds of concentration 10, 5, 2.5. Mu.M and the active oxygen model group were shown in FIG. 7, and the results of the three graphs shown in FIG. 35F-figure 35, respectively, and the results of the samples were plotted in parallel.
As shown in FIG. 7, the sodium glutamate model group has higher active oxygen content than the blank group #### Control group with P < 0.0001vs and P < 0.0001vs sodium glutamate model group) wherein the DCF positive cells were 94.1% and Libertellenone C (at concentrations of 10, 5, 2.5 μm) significantly reduced the DCF positive cell numbers to 34.6%, 45.6%, 54.2%, respectively.
JC-1 staining method for detecting change of cell MMP
SH-SY5Y cells well grown are collected and diluted to a density of 5X 10 5 individual/mL cellsThe suspension was inoculated into a 12-well cell culture plate, placed in a cell incubator and cultured for 24 hours, then the compound represented by formula (1) obtained in example 1 diluted with a DMEM/F12 medium was added so that the final concentration of the compound represented by formula (1) in the well plate was 10, 5, 2.5. Mu.M, respectively, after 4 hours, an appropriate amount of sodium glutamate mother liquor (sodium glutamate diluted with a DMEM/F12 medium) was added so that the final concentration of sodium glutamate in the well plate was 20mM, and a blank group (i.e., a Control group, without adding the compound represented by formula (1) and sodium glutamate) and a sodium glutamate model group (i.e., without adding the compound represented by formula (1), sodium glutamate mother liquor was added so that the final concentration of sodium glutamate in the well plate was 20 mM), after 24 hours, the mitochondrial membrane potential of cells of different administration groups is detected according to a mitochondrial membrane potential detection kit (Solebao) instruction book, the mitochondrial membrane potential results of cells of a blank Control group are shown as a graph A in FIG. 8, the mitochondrial membrane potential results of cells of a sodium glutamate model group are shown as a graph B in FIG. 7, the mitochondrial membrane potential results of cells of a compound shown as a formula (1) with the concentration of 10, 5 and 2.5 mu M are respectively shown as a graph C, D, E in FIG. 8, the cell population proportion of the upper part and the lower part of a transverse line in the A-E graph is counted, the ratio of the upper part to the lower part of the transverse line is calculated, a cylindrical graph is drawn, and the result is shown as a graph F in FIG. 8.
As shown in fig. 8, the lower horizontal line represents the cell population with decreased mitochondrial membrane potential, the upper horizontal line represents the normal cell population, # and P is less than 0.1vs. control group, P is less than 0.01vs. sodium glutamate model group, wherein compared with a blank control group, 20mM sodium glutamate solution can obviously cause the reduction of mitochondrial membrane potential of SH-SY5Y cells, and Libertillenone C can obviously prevent sodium glutamate from causing the reduction of mitochondrial membrane potential of cells at the concentration of 10 and 5 mu M. .
CCK-8 assay to detect cell viability
BV-2: cells BV-2 in logarithmic growth phase were grown at 1.5X10 5 Inoculating into 96-well plate at density of several/mL, adding compound shown in formula (1) obtained in example 1 diluted with DMEM/F12 medium after stable growth for 24 hr to give final concentration of compound shown in formula (1) in the well plate of 40, 20, 10, 5, 2.5 μm, incubating for 1 hr, and settingThe toxicity of the test compound was evaluated according to the survival (%) = OD test/OD control x 100% by adding a proper amount of test solution according to the instructions of CCK-8 reagent, and then measuring the absorbance OD after incubating the test solution with cells at 37 ℃ for 1 hour, and the results of the test compound according to the survival (%) = OD test/OD control x 100% are shown in fig. 9 as a graph a.
As a result, as shown in panel A of FIG. 9, it was revealed that the compound represented by the formula (1) obtained in example 1 was not cytotoxic to BV-2 cells either.
Griess method for detecting NO content in BV-2 cells
Cells BV-2 in logarithmic growth phase were grown at 1.5X10 5 Inoculating the density of each mL into a 96-well plate, after the mixture grows stably for 24 hours, respectively adding the compound shown in the formula (1) obtained in the embodiment 1 diluted by a DMEM/F12 culture medium, so that the final concentration of the compound shown in the formula (1) in the well plate is 10 mu M, 5 mu M and 2.5 mu M respectively, simultaneously, a positive medicine Dexamethasone model group (namely, the compound shown in the formula (1) is not added, the positive medicine Dexamethasone is directly added, so that the final concentration of the positive medicine Dexamethasone in the well plate is 10 mu M), incubating for 1 hour, then, adding LPS again, so that the LPS concentration in the well plate is 1 mu g/mL, setting a control group (namely, the compound shown in the formula (1), the positive medicine Dexamethasone and LPS) and the LPS model group (namely, the compound shown in the formula (1) are not added, and only LPS are added, so that the LPS concentration in the well plate is 1 mu g/mL), incubating for 24 hours, then, taking a clear curve measuring the absorbance value of each well plate is measured according to a standard curve of a contrast of 96 mu L, and measuring the absorbance value of a standard curve, and measuring the absorbance of each well-plate is 100 mu.L; the results of the addition of the compound represented by formula (1) at different concentrations in the blank control group, the positive drug Dexamethasone model group (DXM), the LPS model group are shown in panel B of fig. 9;
according to the figure 9 of the drawings, #### p < 0.0001vs. blank, P < 0.0001vs. LPS modelCompared with a blank control group, LPS (1 mug/mL) can remarkably increase the generation of nitric oxide in BV-2 cells, compared with an LPS model group, libertellenone C has stronger effect of inhibiting the generation of NO at the concentration of 10, 5 and 2.5 mu M, and the inhibition effect is equivalent to that of positive drug Dexamethasone (DXM), so that Libertellenone C has better anti-neuroinflammation effect.
qRT-PCR method for detecting change of relative mRNA level in BV-2 cells
(1) Trizol reagent method (Invitrogen, thermo Fisher Scientific, united States) extracts cellular RNA: cells BV-2 in logarithmic growth phase were grown at 1.5X10 5 The compound of formula (1) obtained in example 1 diluted with DMEM/F12 medium was added after 24 hours of stable growth in 6-well plates at a density of 0/mL, so that the final concentration of the compound of formula (1) in the well plates was 10, 5, 2.5 μm, respectively, incubated for 1 hour, followed by further administration of LPS, so that the LPS concentration in the well plates was 1 μg/mL, and a blank control group (i.e., no compound of formula (1) and LPS) and an LPS model group (i.e., no compound of formula (1) were added, only LPS was added, so that the LPS concentration in the well plates was 1 μg/mL) were set and incubated together for 24 hours. Discarding cell supernatant after finishing treatment, washing with PBS, adding Trizol lysate (1 mL/hole), standing for 10min, adding chloroform 0.2mL, gently mixing upside down, standing at room temperature for 15min, centrifuging at 12000rpm for 15min, collecting supernatant, adding isopropanol with the same volume as that of the supernatant, gently mixing, standing at room temperature for 8min, centrifuging at 12000rpm for 10min, discarding supernatant, washing with 75% ethanol solution (1 mL), centrifuging at 4deg.C, 7500rpm for 5min, discarding supernatant, drying RNA at room temperature or vacuum for 15min, dissolving precipitate (RNA) with enzyme-free sterile water (50 μL/hole), measuring RNA concentration with ultraviolet spectrophotometer (Nanodrop), and storing in-80deg.C refrigerator;
(2) Reverse transcription: taking an enzyme-free EP tube in a super clean bench, adding 2 mug of the RNA obtained by separation (calculated according to concentration), 4 mug of reverse transcription reagent (ABP, USA), adding the total volume to 20 mug by using enzyme-free sterile water, reacting for 10min at 25 ℃ in a metal bath, then reacting for 15min at 42 ℃ and then reacting for 5min at 85 ℃ to obtain target cDNA;
(3) And (3) PCR amplification: a 10. Mu.L system comprising 2. Mu.L of the cDNA obtained above, 2. Mu.L of sterile water, 1. Mu.L of the corresponding primer, 5. Mu. L SYBR Green qPCR Mix (ABP, united States) and a PCR instrument ABIQuantum studio 5 (Thermo Fisher Scientific, USA) was used to perform quantitative RT-PCR with a temperature gradient of 95 ℃ (5 min) & gt 95 ℃ (10 s) & gt 55-60 ℃ (20 s) & gt 72 ℃ (30 s) and a cDNA amplification cycle of 40 times;
(4) Analysis of results: analyzing the amplification result by software QuantStudio Design & Analysis Software, using beta-actin or GAPDH as an internal reference gene, obtaining a relative expression quantity after the expression quantity of a target gene is normalized by the internal reference, comparing and analyzing the relative expression quantity of the target gene in each administration group, and carrying out statistical analysis by using Graphpad Prism 7; the effects of different concentrations of compounds of formula (1) on BV-2 cells iNOS, HO-1, TNF- α, IL-1β, IL-6 and COX-2mRNA levels in the blank control group, LPS model group are shown in FIG. 10;
according to the view of figure 10 there is, #### P＜0.0001， ### the results of the blank control group with P less than 0.001vs, P less than 0.0001, P less than 0.001, P less than 0.01 and P less than 0.1vs LPS model group show that under the action of LPS of 1 mug/mL, mRNA expression level of iNOS, HO-1, TNF-alpha, IL-1 beta, IL-6 and COX-2 in BV-2 cells is obviously increased, and the compound shown in the formula (1) obtained in the embodiment 1 can inhibit the increase of mRNA expression level of inflammatory factors caused by LPS and obviously improve the transcription level of heme oxygenase HO-1 by the treatment of the compound shown in the embodiment 1. Illustrating that the compound of formula (1) obtained in example 1 may exert an anti-inflammatory effect by inhibiting the expression of inflammatory factors such as tumor necrosis factor alpha, interleukin 1 beta and the like
Western Blotting method for detecting changes of relevant protein levels in BV-2 cells
(1) Extraction of BV-2 cell proteins: cells BV-2 in logarithmic growth phase were grown at 1.5X10 5 Inoculating into 6-well plate at density of several mL, after stable growth for 24 hr, adding compound shown in formula (1) obtained in example 1 diluted with DMEM/F12 medium to final concentration of compound shown in formula (1) in the well plate of 10, 5, 2.5 μm, and incubating for 1 hrWhen the LPS was then administered so that the LPS concentration in the well plate was 1. Mu.g/mL, and a blank group (i.e., no compound represented by formula (1) and LPS were added) and an LPS model group (i.e., no compound represented by formula (1) and only LPS were added so that the LPS concentration in the well plate was 1. Mu.g/mL) were set, and incubated together for 24 hours. After the treatment, the supernatant is discarded, PBS is used for lightly washing, a proper amount of protein lysate (ripa: PMSF: cocktail=100:1:1) is added, the mixture is cracked for 30min on ice, shaking is carried out once every 5min, centrifugation is carried out for 10min at the temperature of 4 ℃ and at the speed of 12000rpm after the completion of the cracking, and precipitation is discarded, so that the supernatant is the obtained protein solution;
(2) BCA assay for protein concentration: 1. Mu.L of the protein solution obtained above was taken and 19. Mu.LddH was added 2 Diluting O for 20 times, adding 200 mu L of BCA working solution (A: B=50:1), uniformly mixing, reacting at a constant temperature of 37 ℃ for 30min, detecting the absorbance of each reaction hole at 562nm by using an enzyme-labeling instrument, and calculating the protein concentration according to a standard curve of the protein concentration;
(3) Configuration of polyacrylamide gel: and (3) selecting and configuring polyacrylamide gels with different concentrations according to the molecular weight of the target protein. The experiment adopts 10% SDS-PAGE gel, a kit is rapidly prepared by adopting a Yase PAGE gel, 1.00mm, a lower layer separating gel is firstly prepared according to a formula of a specification, and isopropanol is discarded after the lower layer is gelled and fixed (about 10 min); preparing upper concentrated glue, and preparing a formula according to a specification;
(4) Polyacrylamide gel electrophoresis: after the device is assembled, adding 1 Xelectrophoresis buffer solution into an electrophoresis tank, pulling out a comb, ensuring that the sample loading amount is 10 mug according to the protein concentration measured by BCA, calculating the sample loading volume (V=m/c), adjusting the voltage and time after the sample loading is finished, carrying out electrophoresis at 80V for 30min, and then carrying out electrophoresis at 120V for 60min;
(5) Transferring: immersing a film transfer clamp and an NC film in film transfer liquid, placing gel containing a sample on three layers of filter paper, completely covering the gel with the saturated NC film, taking care of removing bubbles, covering the three layers of filter paper, closing the film transfer clamp, transferring to a film transfer groove, setting the current to 400mA, and constantly transferring to a film for 30min;
(6) Closing: soaking NC membrane transformed with protein in Biyundian rapid sealing solution, and sealing at 4deg.C for 10min;
(7) Washing: the blocking solution was recovered and washed three times with 1 XTBE (10 XTBST diluted 10-fold) for 10min each;
(8) Incubation resistance: cutting corresponding bands according to the molecular weight of the protein, diluting the antibody according to the specification of the antibody, placing the bands containing the target protein into the corresponding diluted antibody, and incubating overnight at 4 ℃ in a shaking table;
(9) Washing: 1 XTBE (10 XTBST diluted 10 times) was washed three times for 10min each;
(10) Secondary antibody incubation: diluting according to the second antibody instruction, placing the corresponding strip in diluted antibody, and incubating for 50min in dark;
(11) Washing: 1 XTBE (10 XTBST diluted 10 times) was washed three times for 10min each;
(12) ECL exposure: ECL reagent (equal volume of mix A, B solution) was uniformly covered on NC membrane and the membrane was placed into a chemiluminescent apparatus for imaging.
As a result, FIG. 11 shows that panel A shows Western immunoblotting of NLRP3, ASC and Caspase-1 with different concentrations of the compound of formula (1), and that the compound of formula (1) obtained in example 1 can significantly inhibit the increase in protein expression levels of NLRP3, ASC and Caspase-1 in BV-2 cells due to LPS, and that panel B-D shows statistics of the values of the protein expression levels of NLRP3, ASC and Caspase-1 with different concentrations of the compound of formula (1) and the ratio of the protein expression levels of NLRP3, ASC and Caspase-1 to the blank ### P＜0.001， # P<Blank 0.1vs. P < 0.01, P < 0.1vs LPS model group). The results indicate that the action mechanism of the compound represented by the formula (1) obtained in example 1 to protect nerve cells may be to inhibit activation of NLRP3 inflammatory bodies.
In conclusion, diterpenoid Libertellenone C can play an anti-neuritic role by inhibiting the activation of NLRP3 inflammatory corpuscles and regulating NF- κB-P65/IκB signal paths, can inhibit the activation of NLRP3 inflammatory corpuscles and regulate NF- κB-P65 signal paths, and finally has remarkable anti-neuritic activity and can be used for preparing medicines for protecting nerve cells.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. An application of diterpenoid compound Libertellenone C shown in formula (1) in preparing a medicine for protecting nerve cells in resisting neurodegenerative diseases.
2. An application of diterpenoid compound Libertellenone C shown in formula (1) in preparing medicine for resisting neuroinflammation direction in resisting neurodegenerative diseases.
3. The use according to claim 1 or 2, wherein the preparation method of diterpenoid compound Libertellenone C represented by formula (1) comprises the steps of:
(1) Inoculating the Phaliota fungus on potato dextrose agar medium, and then culturing at constant temperature to obtain a seed culture medium;
(2) Cutting the seed culture medium into pieces, inoculating the pieces into a rice culture medium, and carrying out fermentation culture;
(3) Extracting the fermentation product obtained in the step (2) by ethanol, and concentrating under reduced pressure to obtain a total extract;
(4) Mixing the total extract with water, extracting with ethyl acetate, and concentrating under reduced pressure to obtain ethyl acetate extract;
(5) And separating the ethyl acetate extract by chromatography to obtain diterpenoid compound Libertellenone C shown in the formula (1).
4. The use according to claim 3, wherein the fungus of the genus sarcodactylis gensis.
5. The use according to claim 3 or 4, wherein in step (1), the conditions of the thermostatic culture comprise: the temperature is 20-30deg.C, and the time is 5-12 days.
6. The use according to claim 3 or 4, wherein in step (2), the conditions of the fermentation culture comprise: the temperature is 20-30deg.C, and the time is 30-40 days.
7. The use according to claim 3, characterized in that in step (2) the solid-to-liquid ratio of rice and water in the rice culture medium is 180-220g:200ml.
8. The use according to claim 3, wherein in step (3) the number of ethanol extractions is 8-15.
9. The use according to claim 3, wherein in step (4) the number of ethyl acetate extractions is 8-13.
10. The use according to claim 3, wherein the specific process of step (5) comprises:
(a) Subjecting the ethyl acetate extract to normal phase column chromatography, wherein the volume ratio is 20:1-0:1 in petroleum ether-ethyl acetate to obtain 5 components Fr.1-Fr.5;
(b) Subjecting the component Fr.5 to ODS column chromatography, and gradient eluting with methanol-water at volume ratio of 20:80-100:0 to obtain 5 components Fr.5.1-Fr.5.5;
(c) Subjecting the component Fr.5.2 to normal phase column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate with the volume ratio of 20:1-0:1 to obtain 5 components Fr.5.2.1-Fr.5.2.5;
(d) And separating and purifying the component Fr.5.2.2 by adopting high performance liquid chromatography to obtain the compound shown in the formula (1).
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