CN114409625B - Keratinone with neuroprotective activity and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of medical microorganisms and medical compounds, and particularly discloses Xylaria nigripes (A)Xylaria nigripes) A compound extracted from rice fermented product is named as Xylarinone (substituted dihydrochromone), and its preparing process and application in pharmacy are disclosed. The compound of the invention has obvious neuroprotective activity, can be applied to the preparation of novel neuroprotective medicaments, and is used for treating but not limited to depression, anxiety and improving cognitive disorder and motor retardation of patients with neurodegenerative diseases. The results of the examples show that the xylenone provided by the invention can obviously improve the survival rate of the PC12 of the damaged nerve cells of the model, and has a good inhibition effect on the apoptosis of the PC12 nerve cells.

Description

Keratinone with neuroprotective activity and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, microorganisms and compounds, in particular to a compound xylenone (substituted dihydrochromone) extracted from a Xylaria nigripes (Xylaria nigripes) rice fermented product, and also discloses a preparation method and application thereof in the pharmaceutical field. The compound has obvious neuroprotective activity and can be applied to the preparation of new neuroprotective drugs.

Background

The natural product refers to the chemical components in the aspects of extracts of animals and plants, or components in the bodies of animals and microorganisms or metabolites thereof, and the like, and mainly comprises proteins, amino acids, polysaccharides, terpenoids, steroids, phenylpropanoids and other compounds (chemical development, 2002,05,405, 407). Because of the advantages of abundant Natural product resources, novel and unique structure, strong biological activity, wide target and unique action mechanism, the Natural product is widely applied to the discovery of active lead compounds and the preparation of new drugs (Journal of Natural Products,1997,60, 52-60).

Higher fungi (highher fungi), also known as Macrofungi (Macrofungi), are one of the important sources of natural products. The investigation shows that the number of fungi in China is at least 10 ten thousand, more than 8000 fungi are reported at present, about 400 fungi are found to have medicinal efficacy through tests, and the number of higher fungi accounts for the most. Some higher fungi are convenient for fermentation culture, and the chemical structure of the produced secondary metabolites changes with the change of external culture conditions, and the expressed biological activities are diversified, so that the higher fungi are regarded as organisms with extremely high creation coefficients (Chinese science foundation, 2007,02, 69-70).

The xylaria fungi have a majority of saprophytic survival modes and a minority of parasitic survival modes, and from tropical zone to temperate zone, the xylaria is widely distributed and usually grows on dead wood or near ant holes, and the fruit xylaria is parasitic on the fallen nuts of the fagaceae plants. Fungi of the genus xylaria play an important ecological role in nature, probably due to their long-term co-evolution with seed plants (Mycological Progress,2019,18, 495-. Xylaria nigripes is a folk medicinal fungus, and chemical components reported about the Xylaria fungi comprise terpenoids, sterols, alkaloids, polyketides and the like, and most of the Xylaria nigripes have biological activities of resisting bacteria, resisting tumors, inhibiting enzymes and the like.

Nowadays, patients with depression, anxiety symptoms or insomnia are more and more, and literature data shows that the current incidence rate of anxiety and depression in China is 5% -6%, the incidence rate of insomnia is 10% -20%, the incidence rate of the elderly is 25%, and the overall incidence rate of mild and moderate psychological disorders is 10% -15% considering the coexistence of anxiety, insomnia and depression (2021, 07, 14-20). Therefore, anti-depression drugs are playing an increasingly important role in combating rising incidence of depression and anxiety, and the potential for the development of the anti-depression and neuroprotective drug market is considerable.

Wulingshen is a drug on the market of Xylaria nigripes, and is widely applied to the treatment of clinical depression and anxiety. Currently, research on the action mechanism of the Xylaria nigripes nerve regulation is mainly extended from related action targets of the previous research on mood disorder diseases (such as on the basis of monoamine transmitter hypothesis), so that researchers cannot well determine the action mechanism and effective chemical components of the Xylaria nigripes nerve regulation medicine. The research on the anti-depression chemical components of Xylaria nigripes and the chemical components with neuroprotective pharmacodynamic activity and the related drug development work of the prior art are very limited.

The inventor of the patent application carries out anti-depression activity screening on the fermentation product of Xylaria nigripes in the earlier stage, and animal experiments show that: the crude extract of the ethyl acetate part of the Xylaria nigripes fermentation liquor can improve the depression-like behavior of mice and improve the expression level of brain-derived neurotrophic factor (BDNF) protein, which indicates that the extract of the ethyl acetate layer of the Xylaria nigripes has anti-depression active ingredients. The invention successfully prepares and separates the Xylaria nigripes secondary metabolite Xylacetone with a novel structure, verifies the neuroprotective activity of the new compound Xylacetone through the neuroprotective activity evaluation experiment, and can be applied to the preparation of new drugs.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a compound, namely carbon ketone (Xylarinone) (substituted dihydrochromone), extracted from a rice fermentation product of Xylaria nigripes (Xylaria nigripes), a preparation method thereof and application thereof in preparing a novel neuroprotective medicament, wherein the medicament prepared by the compound can be used for treating depression, anxiety and improving cognitive disorder and bradykinesia of patients suffering from neurodegenerative diseases.

The compound carbon horn ketone is a compound shown in a structural formula I:

the said formula I carbon horn ketone (Xylarinone) is dihydro chromone compound, 2-propenyl substitution, 5-acetic acid substitution, 7-hydroxy substitution, the systematic name is (7-hydroxy-4-carbonyl-2-propenyl-chromone-5-substitution) acetic acid.

The invention also discloses a preparation method of the novel compound shown in the formula I, which comprises the following steps:

culturing Xylaria nigripes (No. CGBWSHF00611) with rice culture medium, soaking the obtained fermented product in acetone, centrifuging to obtain acetone extractive solution and precipitate, concentrating the acetone extractive solution by evaporation (preferably using rotary evaporator), leaving appropriate amount of water in the extractive solution to obtain acetone layer crude extract, extracting with ethyl acetate, concentrating under reduced pressure to obtain extract, centrifuging to obtain precipitate, repeating the above soaking-centrifuging-concentrating-extracting-concentrating under reduced pressure until the extraction is complete (preferably, the above soaking-centrifuging-concentrating-extracting-concentrating under reduced pressure is carried out for 5 times), mixing to obtain total extract, passing the total extract through 200-300 mesh normal phase silica gel chromatographic column, eluting with petroleum ether/ethyl acetate (1/0,20/1,10/1,5/1,2/1,1/1,0/1, v/v), gradient elution, wherein the solvent elution volume of each gradient is 3 column volumes, 7 crude components are obtained and are sequentially marked as A, B, C, D, E, F and G components, finally, a residual sample on a silica gel column is washed clean, the E component is separated by medium-pressure preparative liquid chromatography, methanol/water mobile phase (20/80,40/60,60/40,80/20,100/0, v/v) is adopted for gradient elution to obtain 5 sub-components which are sequentially marked as E1, E2, E3, E4, E5 components, and the E2 component is subjected to gel chromatography (methanol is used as an eluent), and is separated by preparative liquid chromatography, and a methanol/water system (MeOH/H) is adopted ₂ O,40/60-45/55, v/v, gradient elution for 20min at the flow rate of 4 mL/min), and then the peak is reached at 17.1 min, namely, the carbon keratin compound solution is obtained, and the obtained carbon keratin compound solution is decompressed, concentrated and re-dissolved and volatilized by methanol to obtain carbon keratin crystals.

The invention also discloses application of the compound namely the carbon horn ketone or a pharmaceutically acceptable preparation thereof in preparing a neuroprotective medicament.

Further, the neuroprotective drug is an anti-depression and/or anti-anxiety drug.

In the present invention, the neuroprotective activity of the said compound of formula I is embodied in: the survival rate of the model damaged nerve cell PC12 can be remarkably improved, the inhibition effect on the apoptosis of the PC12 nerve cell is better, and the protective activity on the apoptosis of the PC12 nerve cell induced by oxygen deprivation/reoxygenation (OGD/R) is better than that of the positive control medicament amitriptyline. Therefore, the carbon horn ketone shown in the formula I has potential application in preparing novel antidepressant, anxiolytic and sedative and tranquillizing medicines.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the carbon horn ketone prepared by the invention is a new compound obtained by first preparation, is a substituted dihydrochromone compound, and has a chemical structure completely different from any single-component medicament in the market. The special antidepressant and anxiolytic drug developed based on the compound can avoid the existing drug patents on the market and provide more choices for the market.

2. The invention discloses that the substituted dihydro chromone compound has neuroprotective activity for the first time, is helpful for the research and development field of medicaments to pay attention to the neuroprotective activity of the Xylaria nigripes secondary metabolite, and finds the lead compound with antidepressant and anxiolytic effects.

3. The carbon horn ketone to be protected by the invention has rich sources, can be obtained by means of microbial fermentation, has no chemical pollution in the whole production process, and is green and environment-friendly.

Drawings

FIG. 1 is a hydrogen spectrum (600MHz, CD) of the Ketone prepared in example 1 ₃ OD)；

FIG. 2 is a chart of the carbon spectrum (150MHz, CD) of the Ketone prepared in example 1 ₃ OD)；

FIG. 3 shows the Keratinone prepared in example 1 ¹ H- ¹ H COSY spectrogram;

FIG. 4 is a chart of the HSQC spectra of the charcoal ketones prepared in example 1;

FIG. 5 is a HMBC spectrum of the keratone prepared in example 1;

FIG. 6 is a high resolution-electrospray-mass spectrometry (HR-ESI-MS) spectrum of the carobonone prepared in example 1;

FIG. 7 shows the result of apoptosis (x 200-fold) of PC12 cells of each group in example 2;

FIG. 8 shows the results of the protection of PC12 cell viability by carbon horn ketone in example 2;

FIG. 9 shows the results of the protection of PC12 against apoptosis by xylenone in example 2.

Detailed Description

In order to make the purpose and the inventive content of the present application more clear, the following applicant will clearly and completely describe the technical solution of the present invention with reference to the specific embodiments.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Table 1: laboratory instruments and materials

Example 1 extraction method of cantharidone:

xylaria nigripes (Xylaria nigripes) was collected in 2013 in Yunan province, griffonia, identified by professor of Dayucheng Dai, Beijing university of forestry, and a sample of the strain isolated from fresh fruit bodies (No. CGBWSHF00611) was stored in the institute group of medicinal fungi and national drugs of the institute of medical university of the Central and south national institute, also a strain reported in "Four New Resorcinol derivatives with novel activity from Xylaria nigripes" (Lan-Qing Li, et al. Natural Product Research (2021), DOI:10.1080/14786419.2021.1897591), in particular, line 5 of section 3.2 of this document. Publicly available from the research group.

The preparation method of the strain (No. CGBWSHF00611) is as follows: placing Xylaria nigripes fruiting body in an inoculation box, and sterilizing the surface of mushroom body with 75% alcohol. Longitudinally cutting the middle part of a mushroom stem after the sterilization by a scalpel, cutting 5 squares at a mushroom cap and the mushroom stem by an inoculating shovel, picking a piece of tissue, inoculating the tissue to a PDA (Potato Dextrose Agar) culture medium, culturing at 24 ℃, and obtaining a strain No. CGBWSHF00611 after hypha grows out from the tissue blocks.

The separation and purification process of the carbon horn ketone: the strain cultured by PDA is cut into small pieces and inoculated into rice culture medium, and cultured for 30 days under the dark light condition of 25 ℃. The rice culture medium is prepared by the following steps: 100g of rice and 100ml of water were put into a 500ml conical flask, and sterilized in an autoclave at 121 ℃ for 15 min. Soaking the fermented product in acetone for 24 hr, centrifuging to obtain acetone extractive solution and precipitate, evaporating and concentrating the acetone extractive solution with rotary evaporator, adding water to the extractive solution to obtain acetone layer crude extract, extracting with ethyl acetate to obtain ethyl acetate layer, and concentrating under reduced pressure to obtain extract. Repeating the soaking-centrifuging-concentrating-extracting-decompressing-concentrating process on the precipitate obtained by centrifuging for 5 times, and finally combining the extractum for five times to obtain 182g of total extractum.

Subjecting the total extract to 200-300 mesh normal phase silica gel chromatographic column, eluting with petroleum ether/ethyl acetate mixed solvent (1/0,20/1,10/1,5/1,2/1,1/1,0/1, v/v), sequentially gradient eluting, wherein the solvent elution volume of each gradient is 3 column volumes (the column volume is 2100 cm) ³ ) And finally, washing residual samples on the silica gel column by using methanol to be clean. And (3) rapidly preparing the component E by a medium-pressure liquid chromatograph, wherein an eluent is a methanol/water mobile phase (20/80,40/60,60/40,80/20,100/0, v/v), and sequentially carrying out gradient elution to obtain 5 subcomponents which are sequentially marked as the components E1, E2, E3, E4 and E5. The E2 component is separated by gel chromatographic column (eluent is methanol) and preparative high performance liquid chromatograph under the following conditions: zorbax SB-C18 column (particle size 5 μm, size 9.4 mm. times.150 mm) with methanol/water mobile phase (MeOH/H) ₂ O,40/60-45/55, v/v, gradient elution at 4mL/min flow rate for 20min), followed 17.1 min laterThe peak is the carbon keratin compound solution. The resulting compound solution was concentrated under reduced pressure, redissolved with 0.5mL of methanol, and the solvent was naturally evaporated to give a cantharidone crystal (4.0 mg).

Identification of the structure of Keratinone, shown in FIGS. 1-6, is the hydrogen spectrum (600MHz, CD) of the Keratione crystal prepared in example 1 ₃ OD), carbon spectrum (150MHz, CD) ₃ OD)、 ¹ H- ¹ H COSY spectra, HSQC spectra, HMBC spectra, and high resolution-electrospray-mass spectrometry (HR-ESI-MS) spectra:

keratin crystal, HR-ESI-MS: M/z 285.07288[ M + Na ]] ⁺ (calculated 285.07334) and the molecular formula was determined to be C ₁₄ H ₁₄ O ₅ The unsaturation degree is assumed to be 8. It is composed of ¹³ C NMR and DEPT spectroscopic data (Table 2) gave 14 carbon signals, one CH ₃ (δ _C 17.9), two CH ₂ (δ _C 44.6,42.2), five CH (. delta.)(s) _C 131.1,130.1,115.5,103.4,79.0), six C (with two carbonyl carbons. delta _C 194.1,175.5,166.3,165.1,140.8,113.6)。 ¹ The H NMR spectral data (Table 2) show the proton signals delta on the two benzene rings _H 6.30(1H,d,J＝2.4Hz)，δ _H 6.29(1H, d, J ═ 2.4Hz), the hydrogen atoms on the benzene ring can be judged to be in the meta-position relationship by their coupling constants. Furthermore, from the methine proton signal δ _H 5.89(1H,m)，δ _H 5.68(1H, m), which is presumably a hydrogen atom on a carbon-carbon double bond. ¹ The methylene proton signal delta is also shown on the H NMR spectrum data _H 3.88(2H,dd,J＝10.8Hz)，δ _H 2.56(1H,m)，δ _H 2.70(1H, m), methyl Signal δ _H 1.75(3H, d, J ═ 6.6 Hz). The above data suggests that the compound contains a benzene ring and a tetra-substituted structure exists.

In that ¹ H- ¹ On the H COSY spectrum, a structural fragment, namely-CH can be obtained through hydrogen-hydrogen correlation ₂ (3)-CH(2)-CH(9)-CH(10)-CH ₃ (11) The previous presumption of carbon-carbon double bonds was confirmed(formula I). On HMBC spectra, δ _H 2.56(m, H-3) and C-4 (. delta.) _C 194.1)，C-4a(δ _C 113.6) and delta _H 4.84(m, H-4) and C-4 (. delta.) _C 194.1)，C-8a(δ _C 166.3) to construct an oxygen-containing six-membered ring. Delta. for the preparation of a coating _H 3.88(dd, H-12) and C-13 (. delta.) (delta.) _C 175.5)，C-6(δ _C 115.5)，C-5(δ _C 140.8), correlation of C-4a confirms methylene carbon delta _C 42.2 and carboxyl carbon delta _C 175.5 (formula I), and the structural fragment is attached at the C-5 position of the phenyl ring. The structure of the compound is determined as formula I according to the data, and the compound is a novel compound and is named as carbon horn ketone (Xylarinone).

TABLE 2 method for Keratin ¹ H and ¹³ c NMR data (δ in ppm, J in Hz)

At 600( ¹ H NMR)and 150( ¹³ C NMR)MHz,in methanol-d ₄ 。

Example 2: example 1 Activity assay of the Tacrocan represented by formula I prepared in example 1 for protecting neuronal cells

1. Experimental drugs and reagents

Cell culture fluid RPMI-1640, a gas-permeable cell culture bottle, a 96-well cell culture plate, a 24-well cell culture plate, PC12 cells, fetal bovine serum, trypsin, a CCK8 kit, a penicillin-streptomycin double antibody solution (penicillin working concentration 100U/mL and streptomycin working concentration 0.1mg/L), charcoal keratin, amitriptyline, phosphate buffer PBS (0.03mol/L, pH ═ 7.2), a microporous filter, a syringe, a Hoechst Staining kit, ethanol, DMSO, 4% paraformaldehyde cell fixing solution, Triton X-100, a 50mL centrifuge tube, a 15mL centrifuge tube, an EP tube, a 1mL tip, a 200ul tip, gloves, a mask and the like.

2. Laboratory apparatus

A three-gas incubator, a constant temperature incubator, an enzyme-labeling instrument, a common microscope, a fluorescence microscope, a centrifugal machine, a high-pressure steam sterilization pot, a laser confocal microscope and the like.

3. Experimental methods

3.1 cell treatment

Purchased PC12 cells at 37 ℃ with 5% CO ₂ And (5) performing adaptive culture in an incubator. The culture solution is RPMI-1640 culture solution (containing 5% fetal calf serum and 1% double antibody). At 37 deg.C, 5% CO ₂ Culturing in a saturated humidity incubator, pouring out the culture solution after the cells grow to the bottom of the culture flask, washing for 2 times by PBS, adding trypsin until the final concentration of the trypsin is 0.25%, digesting for 2min, adding RPMI-1640 culture solution to stop digestion, gently blowing, centrifuging for 5min at 1200r/min, re-suspending the cell precipitate, inoculating the cell precipitate in the culture flask, observing and taking a picture under a microscope, and allowing the cells to grow to a monolayer, thus being used for experiments.

3.2 preparation of the solution

Mother solution of working solution: weighing a certain amount of Ketone, dissolving with DMSO, adding PBS to obtain 10% ^-3 mol·L ^-1 The working solution mother liquor of (4), wherein the volume of the added DMSO is 0.5% of the volume of the working solution mother liquor.

Working fluid: will 10 ^-3 mol·L ^-1 (hereinafter mol. L) ^-1 M) was diluted 100-fold with RPMI-1640 medium and prepared into 10-fold aqueous medium ^-5 M working solution, then diluted to 10 in gradient ^-6 、10 ^-7 、10 ^-8 And (4) filtering and subpackaging the working solution of M, and storing at-4 ℃.

The RPMI-1640 culture solution for the amitriptyline as the positive medicament is prepared into 10 ^-6 And (5) M solution.

3.3 establishing, grouping, modeling and dosing of an experimental model:

establishing Oxygen sugar Deprivation-reoxygenation (OGD/R) model, diluting 3.1 treated cells to 10 with RPMI-1640 culture solution ⁴ one/mL cell suspension was inoculated into 96-well plates, 200. mu.L per well and cultured for 24 h. Divided into normal control group (control group), model group, DMSO group, and experimental group (charcoal)Keratin drug group), positive drug amitriptyline group: replacing the whole amount of the normal control group with RPMI-1640 culture solution (containing 5% fetal calf serum), culturing in a constant temperature incubator for 3h, replacing the whole amount of the normal control group with RPMI-1640 culture solution (containing 5% fetal calf serum), and culturing in the constant temperature incubator for 24 h; the total amount of the model group, DMSO group and experimental group is changed into RPMI-1640 sugar-free culture solution in 1% O ₂ ，5％CO ₂ The three-gas incubator is subjected to low-oxygen treatment for 3 hours, DMSO groups are added with DMSO with corresponding doses in working solution, and experimental groups are respectively added with 10 of corresponding pore volumes ^-5 、10 ^-6 、10 ^-7 、10 ^-8 M working solution, amitriptyline group and 10 equal in volume to experimental group ^-6 And (4) M amitriptyline working solution, and then recovering normal oxygen and reoxygenating for 24 hours.

4. Index detection

4.1 morphological Observation of cells

PC12 cells treated according to the method of item 3.3 were then examined under a microscope for cell morphology.

4.2CCK8 method for detecting cell survival rate

After each group of PC12 cells treated according to the method of item 3.3, the total amount was changed to RPMI-1640 culture solution (containing 5% fetal bovine serum), 10. mu.L of CCK8 was added to each well, incubation was performed at 37 ℃ for 3 hours, and absorbance value of each group of cells at 450nm was measured with a microplate reader.

4.3 detection of apoptosis

After each group of PC12 cells was treated according to the method of item 3.3, the cells were gently rinsed 2 times with PBS, 0.3ml of cell fixative was added to each well, and the cells were fixed at room temperature for 45 min. Discarding the solution, washing with PBS 2 times, adding 0.2ml of 0.25% Triton X-100 solution into each well, and treating at room temperature for 10 min. The solution was discarded, washed 2 times with PBS, 0.2mL of Hoechst staining solution (1:400) was added to each well, and the cells were stained for 15min at room temperature. Discarding the solution, washing the cells with PBS 3 times, adding 0.3ml of PBS into each hole, and observing the apoptosis condition under a laser confocal microscope.

5. Statistical treatment

SPSS21.0 software was used for data analysisExperimental data adopted

(mean. + -. standard deviation) as indicated. Differences among groups are compared by using one-factor variance analysis, pairwise comparison is carried out by using an LSD (least squares discriminant) test method when the variances are uniform, and pairwise comparison is carried out by using Tamhane's T2 when the variances are not uniform. P<0.05 indicates that the difference is significant, P<0.01 indicates a very significant difference in difference.

6. Results of the experiment

6.1 morphological characterization of various groups of PC12 cells

The cells of each group of PC12 were observed under an optical microscope, the apoptosis results (200 times) of the PC12 cells of each group are shown in FIG. 7, it can be seen that the cells of the control group had obvious cell bodies and protrusions, the cell morphology was fusiform, the cell morphology of the model group, the DMSO group and the charcoal angle ketone experimental group had some cell bodies and protrusions shrivelled, the cell morphology was circular, and the obvious damage degree was seen.

6.2 survival of PC12 cells in each group

As shown in Table 3, the PC12 cell model group showed significantly lower OD values of cell viability and significantly different survival rates (P) than the control group<0.01); compared with a DMSO group, the model group has no obvious difference; the concentration of the Keratinone drug group was 10 in comparison to the model group ^-6 M and 10 ^-7 The activity OD value and the survival rate of the M cells are obviously increased, and the statistical significance (P) is achieved<0.01), the concentration of the positive drug amitriptyline group is 10 ^-6 M has significant difference (P) compared with the model group<0.01)。

TABLE 3 cell viability OD value and cell viability result of each group of PC12 cells

n＝6

P compared to control group<0.05，**P<0.01; in comparison to the set of models, ^# P<0.05， ^## P<0.01

the results of the protection of PC12 cells from Keratinone are shown in FIG. 8, where P is the ratio of Keratinone to control<0.01; in comparison to the set of models, ^## P<0.01, compared with the control group, the PC12 cell model group has obviously reduced cell survival rate and has significant difference (P)<0.01); compared with a DMSO group, the model group has no obvious difference; the concentration of the Keratinone drug group was 10 in comparison to the model group ^-6 M and 10 ^-7 The survival rate of M cells is obviously increased, and the statistical significance is achieved (P)<0.01) of 10% of Keratinone drug group concentration ^-5 M and 10 ^-8 M has significant difference (P) compared with the model group<0.01) at a concentration of 10 ^-6 The cell survival rate of the drug group of M was higher than that of the positive drug amitriptyline (amitriptyline) group.

6.3 apoptosis of PC12 groups

A fluorescence imaging graph for detecting the apoptosis protection effect of the carbon horn ketone on the PC12 cells by fluorescence imaging shows that the PC12 cells induced by hypoxia/reoxygenation are seriously damaged, chromosomes are concentrated and broken, the cell membrane structure is bubbled to form apoptotic bodies, deep dyeing and brightening appear during Hoechst dyeing, and the content of the apoptotic bodies is 10% ^-6 M and 10 ^-7 M carbon horn ketone shows stronger antioxidation on PC12 cells induced by hypoxia/reoxygenation.

As shown in table 4, the mean fluorescence intensity of PC12 cells in the model group was significantly decreased compared to the control group (P <0.01), the mean fluorescence intensity of PC12 cells in the DMSO group was not significantly different compared to the model group (P >0.05), and the mean fluorescence intensity was significantly increased compared to the model group in the drug group, which was statistically significant (P < 0.05).

TABLE 4 detection results of apoptosis of PC12 cells in each group

n＝6

P <0.05, P <0.01 compared to control; compared to the model group, # P <0.05, # P < 0.01.

The results of the protection of PC12 against apoptosis by cantharidone are shown in FIG. 9, which is compared with the control group<0.01; in comparison to the set of models, ^## P<0.01, the mean fluorescence intensity of PC12 cells in the model group was significantly reduced (P) compared to the control group<0.01), the mean fluorescence intensity of the DMSO group PC12 cells was not significantly different compared with the model group (P)>0.05), the mean fluorescence intensity of the Keratinone drug group is obviously increased compared with that of the model group, and the values have statistical significance (P)<0.05)。

Claims (4)

1. A compound with a structural formula shown as a formula I, which is named as carbon horn ketone;

2. a process for the preparation of a compound according to claim 1, comprising the steps of:

culturing Xylaria nigripes strain No. CGBWSHF00611 by adopting a rice culture medium, soaking the obtained fermentation product by using acetone, carrying out centrifugal separation to obtain an acetone extracting solution and a precipitate, evaporating and concentrating the acetone extracting solution to obtain an acetone layer crude extract, extracting by using ethyl acetate, carrying out reduced pressure concentration to obtain an extract, repeating the soaking-centrifuging-concentrating-extracting-reduced pressure concentrating process on the precipitate obtained by centrifuging until the extraction is complete, combining to obtain a total extract, passing the total extract through a normal phase silica gel chromatographic column with 200 meshes and 300 meshes, and carrying out gradient elution by adopting a petroleum ether/ethyl acetate eluent, wherein the volume ratio of the petroleum ether to the ethyl acetate is sequentially as follows: 1/0,20/1,10/1,5/1,2/1,1/1 and 0/1, wherein the volume of solvent elution of each gradient is 3 column volumes, 7 crude components are obtained, which are sequentially marked as A, B, C, D, E, F and G components, the E component is subjected to medium-pressure preparative liquid chromatography, methanol/water eluent is adopted for gradient elution to obtain 5 sub-components, which are sequentially marked as E1, E2, E3, E4 and E5 components, and during gradient elution, the volume ratio of methanol to water is sequentially: 20/80,40/60,60/40,80/20,100/0 and E2Passing through gel chromatographic column with methanol as eluent, separating by preparative liquid chromatography with methanol/water system MeOH/H ₂ And (3) carrying out gradient elution on O,40/60-45/55, v/v at the flow rate of 4mL/min for 20min, carrying out peak contact at 17.1 min to obtain a carbon ketone compound solution, concentrating the carbon ketone compound solution under reduced pressure, and re-dissolving and volatilizing the carbon ketone compound solution by using methanol to obtain carbon ketone crystals.

3. Use of a compound of claim 1 or a pharmaceutically acceptable formulation thereof in the manufacture of a medicament for neuroprotection.

4. The use according to claim 3, wherein the medicament for neuroprotection is an antidepressant and/or anxiolytic medicament.

Images