Macrocyclic trichothene compound with neuroprotective activity as well as preparation method and application of macrocyclic trichothene compound

Abstract

The invention discloses a macrocyclic trichothene compound with a neuroprotective activity effect as well as a preparation method and application of the macrocyclic trichothene compound. According to the macrocyclic trichothene compound, a new compound Epiroridine acid A is prepared by separating a secondary metabolite derived from a fungus Myrothecium roridine; a cellular level experiment proves that the prepared Epiridine acid A can stimulate a stably transfected cell strain (NGF-293T) of a nerve growth factor to highly express an NGF gene, and can be used for preparing a medicine for neuroprotective treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial applications, and particularly relates to a compound with neuroprotective efficacy, a preparation method thereof, and an application thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and progressive development. Clinically, it is characterized by comprehensive dementia manifestations such as memory impairment, aphasia, apraxia, agnosia, visuospatial skill impairment, executive function disorder, and personality and behavioral changes, and the cause has not been clarified so far. Parkinson's syndrome, also known as paralysis agitans, refers to a clinical syndrome with more than two of the following four main symptoms: 1) resting tremor, 2) muscle rigidity, 3) bradykinesia, 4) loss of postural reflex. Nerve injury refers to the destruction of the integrity of nerve tissue and the impairment of nerve function. However, there is a lack of highly effective and safe drugs with neuroprotective efficacy in the prior art. Summary of the Invention

[0003] The object of the present invention is to provide a compound with neuroprotective efficacy, which is a new compound isolated from the secondary metabolites of the fungus Myrothecium roridum.

[0004] Another object of the present invention is to provide a preparation method of the compound and its application in the preparation of drugs with neuroprotective efficacy.

[0005] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A compound with neuroprotective efficacy, the compound has the following structure:

[0007]

[0008] The preparation method of the compound with neuroprotective efficacy according to the present invention includes the following steps:

[0009] The strain Myrothecium roridum (ATCC 16297) is activated on a PDA plate and then inoculated into a conical flask containing malt culture medium, and cultured on a constant temperature shaker. Then, the cultured seed liquid is aspirated and batch-inoculated into a rice medium, and cultured in a constant temperature environment.

[0010] As a preferred scheme, the strain Myrothecium roridum (ATCC 16297) is activated on a PDA plate and then inoculated into a conical flask containing malt culture medium, and cultured on a constant temperature shaker at 28 °C and 180 rpm for 3 days. Then, 40 mL of the seed liquid after 3 days of culture is aspirated and batch-inoculated into a rice medium, and cultured in a constant temperature environment at 28 °C for 28 days.

[0011] After fermentation is completed, soak with an equal volume of ethyl acetate. Mash the rice fermented mycelium and perform ultrasonic extraction. Concentrate the extract under reduced pressure, and separate it successively using normal-phase silica gel and reversed-phase ODS columns.

[0012] As a preferred option, when using normal-phase silica gel for separation, first perform gradient elution with petroleum ether - ethyl acetate as the elution system; then perform gradient elution with ethyl acetate - methanol as the elution volume to obtain different fractions.

[0013] When using a reversed-phase ODS column for separation, use acetonitrile and water as the mobile phase. Among them, fraction 8 is isocratically eluted with acetonitrile and acidified water with a volume ratio of 35:65 to obtain Epiroridin acid A.

[0014] As an optimized option, the preparation method of the compound with neuroprotective efficacy described above includes the following steps:

[0015] (1) Inoculate the strain Myrothecium roridum on a PDA plate and culture it at 28°C for 2 - 3 days, then inoculate it into a malt liquid medium and culture it with shaking at 120 - 200 rpm for 2 - 5 days; then aspirate 40 mL of the seed liquid after 3 days of culture and batch-inoculate it into a rice medium, and culture it at a constant temperature of 28°C for 28 days.

[0016] (2) After fermentation is completed, soak the rice fermented mycelium with an equal volume of ethyl acetate. Mash the rice fermented mycelium and perform ultrasonic extraction 2 times. The extract is rotary-evaporated at 28°C to recover the solvent to obtain a crude paste extract;

[0017] (3) Perform column chromatography separation of the extract using normal-phase silica gel, and perform gradient elution with petroleum ether - ethyl acetate and ethyl acetate - methanol with different volume ratios, and collect fractions F1 - F9. Among them, fraction F8 is separated by ODS reversed-phase column chromatography and gradient eluted with methanol - water with different volume ratios, and is divided into 15 fractions according to the HPLC analysis of the fractions; among them, fraction 8 is isocratically eluted with acetonitrile and water with a volume ratio of 35∶65 to obtain Epiroridin acid A.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The novel structure compound Epiroridin acid A produced by the fermented fungus Myrothecium roridum in the present invention has significant neuroprotective activity, and this compound and its salts or derivatives can be used to prepare drugs with neuroprotective efficacy. Using the fungus Myrothecium roridum to prepare this novel compound has simple operation, is environmentally friendly, and has low cost. Description of the Drawings

[0020] Figure 1 For the main 1 H- 1 H COSY, HMBC spectra (A) and NOESY spectrum (B) correlation diagrams.

[0021] Figure 2 For the single crystal X-ray diffraction pattern of Epiroridin acid A.

[0022] Figure 3 For 1 1H-NMR spectrum of epiroridin acid A in MeOD (500Hz).

[0023] Figure 4 For 13 13C-NMR spectrum of epiroridin acid A in MeOD (125Hz).

[0024] Figure 5 For the DEPT spectrum of epiroridin acid A.

[0025] Figure 6 For 1 H- 1 H COSY spectrum of epiroridin acid A.

[0026] Figure 7 For the HSQC spectrum of epiroridin acid A.

[0027] Figure 8 For the HMBC spectrum of epiroridin acid A.

[0028] Figure 9 For the NOESY spectrum of epiroridin acid A.

[0029] Figure 10 For the HR-ESI-MS spectrum of epiroridin acid A.

[0030] Figure 11 For the UV spectrum of epiroridin acid A (MeOH).

[0031] Figure 12IR spectrum of epiroridin acid A。

[0032] Figure 13 Cytotoxicity of epiroridin acid A against NGF-293T cells.

[0033] Figure 14 Relative luciferase activity of NGF-293T cells (CON: blank control, CLE: clenbuterol hydrochloride, 1: epiroridin acid A). Specific implementation mode

[0034] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, those skilled in the art's various equivalent modifications of the present invention all fall within the scope defined by the appended claims of this application.

[0035] Example 1 Preparation of compound epiroridin acid A

[0036] 1.1 Fermentation of Myrothecium roridum

[0037] Seed liquid fermentation: Appropriate mycelia were picked from the PDA plate of the strain Myrothecium roridum (ATCC 16297) with an inoculation loop and inoculated into a 1 L conical flask containing 400 mL of ME culture medium. A total of 12 flasks were fermented and cultured on a constant temperature shaker at 28 °C and 180 rpm for 3 days. Then, 40 mL of the seed liquid was aspirated and inoculated into the rice medium. A total of 100 flasks were fermented and cultured in a constant temperature environment at 28 °C for 28 days.

[0038] 1.2 Extraction of secondary metabolites

[0039] After fermentation, it was soaked with an equal volume of ethyl acetate. The rice was mashed and ultrasonically extracted twice. The extract was concentrated under reduced pressure to obtain a total of 146.61 g of extract.

[0040] 1.3 Isolation and purification of secondary metabolites of Myrothecium roridum

[0041] Take 24.14 g of the paste and mix it with 25 g of silica gel for wet sample preparation. Add the sample to a silica gel column containing 250 g of silica gel by dry sample loading method. First, use petroleum ether - ethyl acetate as the elution system for gradient elution, and the elution ratios are: 1:0 (4 L), 50:1 (4 L), 25:1 (4 L), 10:1 (4 L), 5:1 (4 L), 2:1 (4 L), 1:1 (4 L), 0:1 (4 L); then use ethyl acetate - methanol as the elution system for gradient elution, and the elution ratios are: 50:1 (2.4 L), 20:1 (2.4 L), 10:1 (2.4 L), 5:1 (2.4 L), 2:1 (4 L), 1:1 (2.4 L), 0:1 (4.5 L), and a total of 15 fractions (Fr.G1 - 15) are obtained.

[0042] Take fraction Fr.G8 of 0:1 (4 L) from the petroleum ether - ethyl acetate elution system and fraction Fr.G9 of 50:1 (2.4 L) from the ethyl acetate - methanol elution system, and prepare Epiroridin acid A by semi - preparative high - performance liquid chromatography. The preparation conditions are: isocratic elution with 35% acetonitrile: 65% acidic water (1‰ formic acid water). Stationary phase: Fisher Wharton XBridge 5 - μm C18 column (10×250 mm); flow rate: 2 mL / min; detection wavelength: 254 nm.

[0043] Example 2 Structure Analysis of Epiroridin acid A

[0044] Epiroridin acid A is a white needle - shaped crystal. HR - ESI - MS m / z: 561.2356 [M - H] - (C 29 H 37 O 11 Calculated value 561.2341), and the molecular formula is C 29 H 38 O 11 , and the degree of unsaturation is 11. By analyzing 1 1H - NMR (Table 1) data, it can be seen that this compound contains 3 methyl signals (δ H 0.85, s, 14 - H3; 1.10, d, J = 6.8 Hz, 12'-H3; 1.12, d J = 6.4 Hz, 14'-H3), 2 characteristic hydrogen signals of exocyclic epoxides (δ H 2.88, d, J = 3.9 Hz; 3.08, d, J = 3.9 Hz, 13 - H2), 5 olefinic proton signals (δ H6.76, d, J = 4.7 Hz, 10-H; 6.15, dd, J = 15.4, 3.0 Hz, 7'-H; 7.64, dd, J = 15.4, 11.7 Hz, 8'-H; 6.79, d, J = 11.5 Hz, 9'-H; 5.78, d, J = 11.7, 11.1 Hz, 10'-H). From these signal information, it can be speculated that this compound is a roridin analogue. From the molecular formula, it can be seen that compared with the known compound roridin A, this compound has 2 fewer H atoms, 2 more O atoms, and 1 more degree of unsaturation. Combining the analysis 13 The 13C-NMR (Table 1) data can determine that this compound contains 29 carbon units, and further determine that this compound contains 3 methyl signals (δ C 8.0, C-14; 15.1, C-12'; 18.3, C-14'). Combining with the analysis of the DEPT spectrum, the difference from roridin A is that this compound has 1 fewer methyl signal and 1 more carbonyl carbon signal (δ C 170.0, C-16). Thus, it can be speculated that there may be a carboxyl substitution at the 16th position of roridin A in this compound. By detailed interpretation of the 2D NMR spectrum of this compound, the planar structure can be constructed. Based on 1 H- 1 H COSY data, it can be seen that there are correlation signals between H-2 and H-3; H-3 and H-4; H-7 and H-8; H-10 and H-11; H-3' and H-2', H3-12', H-4'; H-4' and H-5'; H-6' and H-7'; H-7' and H-8'; H-8' and H-9'; H-9' and H-10'; H-13' and H3-14' ( Figure 1 ). Thus, the structure of the macrocyclic trichothecene is correlated. According to the HMBC correlations from H-8, H-10 to C-16, the connection between C-16 and C-9 is clarified ( Figure 1 ). Therefore, the planar structure of the compound is constructed as a macrocyclic trichothecene compound. Figures 3 to 12 . By analyzing the NOESY of the compound, it can be seen that there is a correlation between H-2 and H-14, and a correlation between H-14 and H-13. Thus, it is judged that H-2 / H-13 / H-14 are in the same orientation, designated as β. Because there are correlations between H-4 and H-11, H-15, and there are no correlations between H-11, H-15 and H-2, H-13, H-14, it is judged that H-4 and H-11, H-15 are in the same orientation, which is α. Also, because there is a correlation between H-2' and H-2, it is judged that H-2' and H-2 are in the same orientation, which is β. In addition, according to the hydrogen at C-2' position δ HFrom the coupling constant of 4.07 (J = 3.9 Hz), it indicates that the orientations of H-2' and H-3' are the same, that is, 12'-CH 3 and 2'-OH are in the same orientation. There is a correlation between H-6' and H-12', indicating that the orientations of H-6' and 12'-CH 3 are consistent; there is no obvious correlation between H-13' and the hydrogens at other positions. Thus, the relative configuration of the compound can be determined as 2R,4R,5S,6R,11R,12S,2'S,3'R,6'R 13'R or 2R,4R,5S,6R,11R,12S,2'S,3'R,6'R,13'S or 2S,4S,5R,6S,11S,12R,2'R,3'S,6'S,13'R or 2S,4S,5R,6S,11S,12R,2'R,3'S,6'S,13'S. Finally, the absolute configuration of Epiroridin acid A is determined as 2R,4R,5S,6R,11R,12S,2'S,3'R,6'R 13'R by single crystal X-ray diffraction (the data is stored in the Cambridge Crystallographic Data Centre no. CCDC - 2357113)( Figure 2 )

[0045] Table 1. 1 H NMR and 13 C-NMR Data of Epiroridin acid A (δ in ppm, J in Hz)

[0046]

[0047]

[0048] Recorded at a Bruker AVANCE-500 in MeOD.

[0049] Example 3 Evaluation of Neuroprotective Activity

[0050] 1. Experimental Cells

[0051] HEK-293T cells were provided by the Biomedical Materials Engineering Research Center of Sichuan University. Method for obtaining NGF-293T cells: Insert the NGF (nerve growth factor) promoter sequence and the luciferase reporter gene sequence into the multiple cloning site of the pUC57 vector to obtain the recombinant vector NGF-Luc-pUC57. Transfect the recombinant vector NGF-Luc-pUC57 into HEK-293T cells to obtain NGF-293T cells, which can stably express luciferase.

[0052] 2. Main Experimental Reagents and Consumables

[0053] NGF-Elisa kit (Ruixin Bio, RX302374R), protein extraction kit (Thermo Fisher Scientific, 23227), CCK-8 kit (biosharp), total RNA extraction kit (Solarbio, R1200-100T), Steady-Lumi TM Firefly luciferase reporter gene detection kit (Beyotime, RG058S), Firefly luciferase reporter gene cell lysis buffer (Beyotime, RG126M), MEM-ALPHA medium (VIVA Cell, 220808), D-hanks solution (Procell), high-glucose medium (Procell, WH2622D221), PBS (Saiwei'er), fetal bovine serum (Sijiqing, 11011-8611), 1% double antibody (Thermo Fisher Scientific, 15140122), 0.25% trypsin (Cytiva, SH30042.01), Clenbuterol hydrochloride (Maclin, C805465-10mg). Epiroridin acid A was prepared in our laboratory.

[0054] 3. Cytotoxicity of the drug Epiroridin acid A on NGF-293T cells

[0055] Inoculate the NGF-293T cell suspension in a 96-well plate, 100 μL / well, and control the number of cells to be 7×10 3 -1×10 4 / well, and culture in a cell incubator at 37°C and 5% CO 2 for 18 h. Add 100 μL of Epiroridin acid A samples with final concentrations of 0.1, 0.5, 1, 2.5, 5, and 10 μM to each well in the dosing group. After incubating for 24 h, add 10 μL of CCK-8 solution to each well. After incubating for 2 h, measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm. In the control group, the positive drug clenbuterol was used to replace the Epiroridin acid A solution, and the others were the same as the dosing group; in the blank group, there were no cells and no Epiroridin acid A solution was added. Calculate the cell survival rate according to the formula: cell survival rate = (dosing group - control group) / (blank group - control group) × 100%. The results are as follows Figure 13 , doses of 0.1, 0.2, 0.5, 1, and 2.5 μM promoted the proliferation of NGF-293T first, and inhibitory effects occurred when the concentration was higher than 10 μM. Therefore, 2.5 μM was selected as the basic dosing concentration.

[0056] 4. Luciferase activity detection

[0057] Take cells at passages P2 - P3. After trypsin digestion, add an appropriate amount of culture medium and mix well. Then, evenly seed the cells into a 6 - well plate at a density of 150,000 - 200,000 cells per well, with 2 mL per well, and place it in a CO 2 incubator. After the cells adhere to the wall, use a pipette gun to aspirate the old culture medium, and then add 2 mL of a 2.5 M drug solution (Epiroridin acid A) to each well. Incubate in the incubator for 24 h. After the incubation ends, aspirate the old culture medium, add reporter gene cell lysis buffer. After lysing for 15 min, centrifuge at 12,000 rpm for 5 min, and take the supernatant for subsequent determination.

[0058] Take the supernatant obtained in the previous step and add 20 μL to a black 96 - well plate, with 3 replicates per group. Then add 100 μL of the thawed Steady - Lumi TM luciferase detection reagent, mix well, and incubate in the dark at room temperature for 5 min to stabilize the luminescence signal. Finally, perform chemiluminescence detection on a microplate reader.

[0059] 5. BCA protein content determination

[0060] Prepare the BCA protein standard solution according to the instructions, and prepare the working solution (Solution A: Solution B = 50:1) with the required volume according to the BCA protein concentration detection kit, paying attention to light - proof operation. Add 25 μL of the standard solution per well and 5 μL of the sample per well to a 96 - well plate. Then add 20 μL of MPER solution to the sample wells to make up the volume. After that, add 200 μL of the prepared BCA working solution to the standard wells and sample wells respectively, and incubate at 37 °C in the dark for 30 min. Measure the OD value at 562 nm on a multifunctional microplate reader. Relative luciferase activity = (luciferase activity in the drug - treated group / protein concentration in the drug - treated group) / (luciferase activity in the blank group / protein concentration in the blank group) × 100%.

[0061] Figure 14 The relative luciferase activity after drug intervention is shown. When the drug at a concentration of 1 μM is co - cultured with NGF - 293T cells, compared with the positive drug clenbuterol hydrochloride, Epiroridin acid A can significantly stimulate the high - expression of the NGF gene in NGF - 293T cells.

[0062] The above - mentioned is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A macrocyclic trichothecene compound having neuroprotective efficacy, characterized in that: The structural formula of the compound is as follows:

2. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: The strain Myrothecium roridum (ATCC 16297) was activated with a PDA plate and inoculated into a conical flask filled with malt culture medium, and cultured on a constant temperature shaker. Then, the seed liquid after culture was aspirated and inoculated into rice culture medium in batches, and cultured in a constant temperature environment.

3. The method for preparing the compound according to claim 2, characterized in that: The following steps are involved: The strain Myrothecium roridum (ATCC 16297) was activated with a PDA plate and inoculated into a conical flask containing malt culture medium, and cultured at 28°C, 180 rpm constant temperature shaker for 3 days. Then, 40 mL of the seed liquid after 3 days of culture was aspirated and inoculated into rice culture medium in batches, and cultured at 28°C for 28 days.

4. The preparation method according to claim 2 or 3, characterized in that: The compound Epiroridin acid A was extracted by the following method: after fermentation, the fermented rice mycelium was soaked with an equal volume of ethyl acetate, crushed and then ultrasonically extracted, the extract was concentrated under reduced pressure, and separated using normal phase silica gel and reverse phase ODS columns in turn.

5. The preparation method according to claim 4, characterized in that: When normal phase silica gel is used for separation, gradient elution is first performed using petroleum ether-ethyl acetate as the elution system; then gradient elution is performed using ethyl acetate-methanol as the elution volume to obtain different fractions.

6. The preparation method according to claim 4, characterized in that: When a reversed phase ODS column is used for separation, acetonitrile and water are used as mobile phases, wherein fraction 8 is isocratically eluted with acetonitrile and acid water in a volume ratio of 35:65 to obtain Epiroridin acidA.

7. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) Fermentation of Myrothecium roridum The strain Myrothecium roridum (ATCC 16297) was activated with a PDA plate and inoculated into a conical flask containing malt culture medium, and cultured at 28°C, 180 rpm constant temperature shaker for 3 days. Then, 40 mL of the seed liquid after 3 days of culture was aspirated and inoculated into rice culture medium in batches, and cultured at 28°C for 28 days. (2) Extraction of secondary metabolites After fermentation, the rice was soaked with an equal volume of ethyl acetate, crushed and ultrasonically extracted for multiple times, and the extract was concentrated under reduced pressure to obtain an extract; (3) Isolation and purification of secondary metabolites of Myrothecium roridum The paste prepared in step (2) was mixed with silica gel for wet sample mixing, and the sample was added to the silica gel column by dry sample loading method. Gradient elution was first performed using petroleum ether-ethyl acetate as the elution system, and the elution volume ratio was: 1:0 (4 L), 50:1 (4 L), 25:1 (4 L), 10:1 (4 L), 5:1 (4 L), 2:1 (4 L), 1:1 (4 L), 0:1 (4 L); and then gradient elution was performed using ethyl acetate-methanol as the elution volume, and the elution ratio was: 50:1 (2.4 L), 20:1 (2.4 L), 10:1 (2.4 L), 5:1 (2.4 L), 2:1 (4 L), 1:1 (2.4 L), 0:1 (4.5 L), and a total of 15 fractions Fr.G1-15 were obtained; The fraction Fr.G8 was separated by semi-preparative HPLC. The preparation conditions were as follows: acetonitrile: water containing 1‰ formic acid in a volume ratio of 35:65, isocratic elution; stationary phase: Fisher Wharton XBridge 5-μm C18 column, size 10×250mm; flow rate: 2mL / min; detection wavelength: 254nm.

8. Use of the compound according to claim 1 and its salt or derivative in the preparation of neuroprotective drugs.

9. Use of the compound according to claim 1 and its salt or derivative in the preparation of medicines for treating Alzheimer's disease and Parkinson's disease.

10. The use according to claim 8 or 9, characterized in that: The salts include inorganic acid salts or organic acid salts.

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