CN114573537B - Compound with anti-neurodegenerative change activity, preparation method and application thereof - Google Patents
Compound with anti-neurodegenerative change activity, preparation method and application thereof Download PDFInfo
- Publication number
- CN114573537B CN114573537B CN202210197180.1A CN202210197180A CN114573537B CN 114573537 B CN114573537 B CN 114573537B CN 202210197180 A CN202210197180 A CN 202210197180A CN 114573537 B CN114573537 B CN 114573537B
- Authority
- CN
- China
- Prior art keywords
- compound
- component
- obtaining
- penicillium
- neurodegenerative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/80—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/753—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups
- C07C49/755—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups a keto group being part of a condensed ring system with two or three rings, at least one ring being a six-membered aromatic ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/38—Cyclopentanone- or cyclopentadione-containing products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/36—Ortho- or ortho- and peri-condensed systems containing three rings containing eight-membered rings
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application discloses a compound with anti-neurodegenerative change activity, a preparation method and application thereof. The compound with anti-neurodegenerative change activity can be produced by adopting a mode of co-culturing two strains of the alternaria brassicae and the penicillium, and the capability of synthesizing secondary metabolites of microorganisms is excavated as much as possible by changing a culture medium, a culture condition, a mixed culture proportion and other research methods, so that the amplification culture can be carried out on the basis of repeatable and sustainable development of the process. The compound has neuroprotective activity on cells with neurodegenerative change diseases, and shows better protective effect than a positive medicine tert-butylhydroquinone (TBHQ).
Description
Technical Field
The application relates to the technical field of medicines, in particular to a compound with anti-neurodegenerative change activity, a preparation method and application thereof.
Background
Neurodegenerative diseases are caused by the loss of neurons and/or their myelin sheath, worsening over time, and presenting with dysfunction. Neurodegenerative disorders affect many people worldwide, such as Alzheimer's disease and Parkinson's disease, and the severity and harmfulness of their symptoms have a tremendous impact on the quality of life of patients and their families.
The clinical treatment of neurodegenerative diseases mainly comprises surgical treatment and chemical drug treatment. Surgical treatment is limited in its wide use in the treatment of neurodegenerative disorders because of the high recurrence rate of the disease after surgery. In the aspect of drug treatment of neurodegenerative change diseases, the existing drugs for treating Alzheimer disease only can delay deepening of pathological symptoms, can not effectively reverse or prevent the occurrence process of the diseases, can generate drug resistance and adverse reaction with different degrees after long-term administration, and easily cause disease recurrence after drug withdrawal; in addition, medicines for treating Parkinson's disease such as L-DOPA can cause abdominal distension and gastrointestinal reaction with different degrees at the initial stage of taking, and long-term taking of L-DOPA easily causes side effects such as illusion or clinically unpredictable switching phenomenon, and the like, so that the daily life of a patient is greatly influenced. Therefore, drug treatment of neurodegenerative diseases remains a clinical challenge to be addressed.
At present, medicines for resisting neurodegenerative change diseases are mainly extracted and separated from natural products such as animal and plant medicines, the growth cycle is long, the raw material acquisition difficulty is high, and meanwhile, a large amount of raw material requirements easily cause resource deficiency of different degrees and even cause exhaustion. In addition, the active secondary metabolite components and contents thereof are very different due to the difference in the place of production and the animal and plant drugs occurring in different seasons, resulting in difficulty in reproduction of the acquisition of the active components.
Since the 20 th century, scientists have conducted extensive research on secondary metabolites of microorganisms, and have successively discovered a number of small molecules with biological activities, including terpenes, flavonoids, alkaloids, etc., while diterpenoids discovered therefrom have significant anti-inflammatory, antioxidant, antitumor, neuroprotective, etc. activities. Under natural environment, antagonism and synergy among different microorganisms are very common, and compared with the silencing phenomenon of a biosynthetic gene cluster of a single microorganism, the co-cultured microorganism can generate various and rich secondary metabolites through the synergy of different metabolic enzymes or metabolites. Therefore, the co-culture method is adopted to optimize culture conditions, metabolic products and biological activities of fermentation products of co-culture fungi are mined, and the method has extremely important significance for developing novel medicines for treating neurodegenerative diseases.
Disclosure of Invention
In view of the above, it is an object of the present application to provide a compound having an anti-neurodegenerative change activity, a preparation method and an application thereof, so as to solve one of the above technical problems to some extent.
In a first aspect, embodiments herein disclose compounds having anti-neurodegenerative altering activity, including, for example
And/or +.>
A compound shown; or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.
In the examples of the present application, the compound of formula I includes, for example
And/or
A compound shown;
the chemical formula IIThe composition comprises
And/or +.>
The compounds shown.
In a second aspect, embodiments of the present application disclose a formulation for treating neurodegenerative disorders, wherein the formulation comprises a compound of the first aspect and a pharmaceutically acceptable adjuvant.
The pharmaceutically acceptable auxiliary materials comprise at least one of diluents, carriers and excipients.
"excipient" refers to a pharmaceutically acceptable material, composition or vehicle that participates in imparting a form or consistency to a pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when mixed so as to avoid interactions that would greatly reduce the efficacy of the compounds of the invention when administered to a patient and avoid interactions that would result in the pharmaceutical composition not being pharmaceutically acceptable. In addition, each excipient must be of sufficiently high purity to be pharmaceutically acceptable.
Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form selected. In addition, suitable pharmaceutically acceptable excipients may be selected for the particular function they may use in the composition. For example, certain pharmaceutically acceptable excipients may be selected because of their ability to facilitate the production of a uniform dosage form. Certain pharmaceutically acceptable excipients may be selected because of their ability to produce stable dosage forms. Certain pharmaceutically acceptable excipients may be selected because they facilitate carrying or transporting one or more compounds of the invention once administered to a patient from one organ or body part to another organ or body part. Certain pharmaceutically acceptable excipients may be selected because of their ability to enhance patient compliance.
Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, wetting agents, chelating agents, plasticizers, tackifiers, antioxidants, preservatives, stabilizers, surfactants, and buffers. As will be appreciated by those skilled in the art, certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much excipient is present in the formulation and what other ingredients are present in the formulation.
The formulations provided by embodiments of the present application may be in a form suitable for use in the following manner: oral use (e.g., as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets and cachets), topical use (e.g., as a cream, ointment, emulsion, solution, paste, spray, foam and gel), transdermal administration (e.g., by transdermal patch), administration by inhalation (e.g., as a dry powder, aerosol, suspension and solution), administration by insufflation (e.g., as a fine powder) or parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular administration, or as a suppository for rectal administration).
In a third aspect, embodiments herein disclose a method for preparing a compound of the first aspect, comprising the steps of:
respectively obtaining activated colonies containing alternaria brassicae and penicillium;
inoculating the bacterial colony to a fermentation medium for fermentation to obtain a fermentation liquid; and
extracting and separating the fermentation liquor to obtain the compound;
wherein, the brassica alternaria alternata, latin name Alternaria brassicicola, is from China center for agricultural microbiological culture collection center, deposit number: ACCC 37296;
penicillium graininess, latin name Penicillium granulatum, from China center for collection of marine microorganism strains, accession number: MCCC 3a00475.
In the examples herein, activated colonies containing Alternaria brassicae and Penicillium are obtained by inoculating the strain into PDA medium and culturing at 28deg.C.
In embodiments of the present application, the fermentation medium comprises 50wt% rice.
In an embodiment of the present application, the step of extracting and separating includes:
obtaining a crude extract, wherein the crude extract is obtained by extracting the fermentation liquor with ethyl acetate;
obtaining a fine extract, wherein the fine extract is obtained by extracting the crude extract sequentially by petroleum ether, dichloromethane and ethyl acetate; and
the compound is obtained by purifying the refined extract through normal phase silica gel chromatography, reverse phase silica gel chromatography and high performance preparation chromatography.
In an embodiment of the present application, the step of obtaining the compound specifically includes:
obtaining a first component, wherein the first component is obtained by loading the refined extract to a normal phase silica gel column and collecting eluent;
obtaining a second component, wherein the second component is obtained by loading the first component to a reverse phase silica gel column and collecting eluent;
and (3) obtaining a third component, wherein the third component is obtained by loading the second component to a high performance liquid chromatography column and collecting eluent, and the third component comprises a compound shown as a formula I and/or a formula II.
In a fourth aspect, embodiments of the present application disclose the use of a compound according to the first aspect, or a compound prepared by a preparation method according to the second aspect, in the preparation of a medicament for treating a neurodegenerative disease.
Compared with the prior art, the application has the following beneficial effects:
the present application relates to compounds having activity against neurodegenerative changes, methods of preparation and uses thereof. By adopting a strategy of co-culturing two strains, research methods such as culture medium, culture conditions, mixed culture proportion and the like are changed, epigenetic regulation is carried out on silent biosynthesis genes, the capability of synthesizing secondary metabolites of microorganisms is mined as much as possible, and the amplification culture can be carried out on the basis of repeatable and sustainable development of the process, so that the resource waste is avoided.
Second, the compounds provided herein have neuroprotective activity against cells of neurodegenerative disease models and exhibit better protective effect than the positive drug TBHQ (terbutylhydroquinone) at a concentration of 25 μm. The compound can be expected to develop into medicines related to treating neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.
In addition, the frozen strains are revived and passaged according to an experimental scheme, and the frozen strains are cultured according to the optimized culture conditions, so that infinite amplification fermentation can be realized, the yield of the compound can be purposefully regulated by regulating and controlling the total amount of the fermented product, and finally, the target product with the function of resisting the neurodegenerative change disease is obtained.
Drawings
FIG. 1 is a graph showing the comparison of the calculated ECD values with the calculated ECD values for the compounds of formula II provided in the examples of the present application.
FIG. 2 is a single crystal structure of the compound of formula I-2 provided in the examples of the present application.
FIG. 3 is a graph showing the cytotoxic effect of the compounds and positive drug TBHQ on SH-SY5Y cells provided in the examples of the present application.
FIG. 4 shows the results of the examples of the present application for the compounds and the positive drug TBHQ vs H 2 O 2 Protection of SH-SY5Y cells induced injury.
FIG. 5 shows the results of the examples of the present application for the compounds and the positive drug TBHQ vs H 2 O 2 Photographs of SH-SY5Y cell action effect of induced injury.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
Preparation of the Compound of formula I/formula II
1. Strain
The compounds shown in the formula I or the formula II disclosed in the examples are obtained by fungal fermentation culture to extract and separate secondary metabolites.
The species referred to in the examples of the present application are derived from the following sources:
alternaria brassicae, latin name Alternaria brassicicola, is derived from China center for type culture Collection of microorganisms, accession number: ACCC 37296;
penicillium graininess, latin name Penicillium granulatum, from China center for collection of marine microorganism strains, accession number: MCCC 3a00475.
2. Strain activation
The culture medium is prepared according to the following formulation proportion, and the operation steps are as follows:
(1) Pouring the weighed PDA into a blue cap bottle, adjusting the pH to 7.0 by NaOH, and placing the bottle into a sterilizing pot to set the temperature to 121 ℃ for 30min.
(2) Sterilizing with ultraviolet for 30min, cooling to about 50deg.C, adding antibiotic and histone deacetylase inhibitor (nicotinamide), and pouring into plate.
(3) Inoculating Alternaria brassicae (Alternaria brassicicola) ACCC 37296 and Penicillium graininosum (Penicillium granulatum) MCC 3A00475 into the culture medium according to the weight ratio of 5:1 by plate streaking method after the culture medium is completely coagulated for 15min, and culturing at 28deg.C for 1 week to obtain plate colony.
3. Fermentation
The plate colonies obtained by the above culture were diced to obtain agar blocks containing colonies, and the agar blocks were inoculated into a sterilized fermentation medium at an inoculation ratio of 6wt% (based on the weight of the fermentation medium), and air oxygen content was fermented at normal pressure for 28 days to obtain a fermentation broth. The fermentation medium is 50wt% (mass percent) of rice water solution, and the rice can be soaked in advance and crushed;
4. extraction and separation
(1) Obtaining coarse extract and fine extract
Extracting the fermentation liquor with 95% ethanol, and recovering the solvent from the extraction liquor under reduced pressure to obtain 389g of crude extract; then 5L of hot water with the temperature of 50 ℃ is used for suspension, equal volume petroleum ether, dichloromethane and ethyl acetate are used for extraction in sequence, and then the extraction part of ethyl acetate is used for recovering the solvent, thus 112g of refined extract is obtained.
(2) Obtaining a first component
Loading 112g of the refined extract on a normal phase silica gel chromatographic (60-80 meshes) column, and eluting with petroleum ether-acetone gradient (70% petroleum ether-20% petroleum ether) to obtain 10 fractions, which are named as Fr.1-Fr.10 in sequence;
loading the fraction Fr.6 on a normal phase silica gel column again, and performing gradient elution by petroleum ether-ethyl acetate (65% petroleum ether-25% petroleum ether) to obtain 9 fractions, which are named Fr.6.1-Fr.6.9 in sequence; wherein fr.6.7 is the first component.
(3) Obtaining a second component
Loading the first component Fr6.7 to a reverse phase silica gel column, and performing gradient elution by methanol-water (70% methanol-30% petroleum ether) to obtain 6 fractions which are named Fr.6.7.1-Fr.6.7.6 in sequence; wherein fr.6.7.1 is the second component.
(4) Obtaining a third component
The second component fractions fr.6.7.1 were each loaded onto a reverse phase high performance chromatography preparation column (Acclaim TM Trinity Q1 LC, cat# 083244; mobile phase: acetonitrile: water=30:70, 2 ml/min); the compounds shown in the formula I and the formula II can be respectively obtained;
if the compounds shown in the formula I and the formula II are respectively loaded to chiral chromatographic columns for preparation (a large xylonite CHIRALPAK IG chromatographic column, mobile phase: acetonitrile: water=28:72, 2 ml/min), the compounds shown in the formula I-1\I-2 can be respectively obtained; and a compound shown as a formula II-1\formula II-2.
Structural identification of Compounds of formula I/II
The absolute configuration of the compounds shown in the formula I and the formula II is obtained by Nuclear Magnetic Resonance (NMR), electron Circular Dichroism (ECD) and X-single crystal diffraction (X-Ray) methods respectively.
1. Nuclear magnetic resonance
A compound of formula I-1: colorless blocky crystals; 1 h and 13 c NMR data are shown in Table 1.
A compound of formula II-1: white powder; high resolution electrospray ionization mass spectrometry (HRESIMS) [ M+Na ]] + m/z 353.1724 (calculated value C 20 H 26 O 4 Na,353.1723)；
UV(CH 3 OH)λ max (logε)＝234(5.93)nm；ECDλ max (Δε)212(-4.59),236(+6.04)nm；IR(KBr)ν max 3455,2927,2872,1761,1698,1457,1383cm –1 ； 1 H and 13 c NMR data are shown in Table 1.
A compound of formula I-2: colorless blocky crystals; high resolution electrospray ionization mass spectrometry (HRESIMS) [ M+Na ]] + m/z 371.2200 (calculated value C 21 H 32 O 4 Na,371.2193)；
UV(CH 3 OH)λ max (logε)＝207(5.79),243(5.93)nm；IR(KBr)ν max 3433,2960,2925,1678,1629,1456,1382cm –1 ； 1 H and 13 c NMR data are shown in Table 2.
TABLE 1
a Measured in CD 3 OD
TABLE 2
a Measured in CDCl 3
2. The absolute configuration of compound 1 was verified by means of a method of calculating Electron Circular Dichroism (ECD).
Results: the experimental tests show that the Cotton effect of Electronic Circular Dichroism (ECD) is consistent with the calculated values, see FIG. 1.
3. X-single crystal diffraction (X-Ray) analysis
By X-Ray analysis of copper targets of the compounds of formula I-2.
Results: the absolute configuration, crystal number, of the compound of formula I-2 was determined: CCDC214277, see fig. 2.
The final compound was determined to be
The compounds shown.
Cytotoxicity study
The cell viability was assessed by CCK-8 assay, which comprises the steps of:
SH-SY5Y cells or H 2 O 2 (350. Mu.M) injured SH-SY5Y cells were seeded on 96-well plates for 24 hours;
mu.L of 10% (v/v) CCK-8 solution was added and the cells were placed in a dark incubator for 2 hours;
absorbance (OD) values were recorded at 450 nm.
Cell viability was calculated by the following formula: cell viability% = (experimental OD-blank OD)/(normal OD-blank OD) ×100%; the results of cell viability were obtained from the mean of the standard deviations (n=3), wherein,
blank group: adding only the culture medium group;
normal group (Normal Control): untreated group;
experiment group 1: compound 1 treatment groups with different concentration gradients were added;
experiment group 2: adding compound 2 treatment groups with different concentration gradients;
experiment group 3: compound 3 treatment groups with different concentration gradients were added;
experiment group 4: adding positive medicine tert-butylhydroquinone (TBHQ) treatment groups with different concentrations;
wherein, the compounds 1, 2 and 3 are respectively shown in the formulas II-1, I-1 and I-2.
Results: as shown in FIG. 3, through analysis of the CCK-8 method data, compounds 1-3 have no obvious cytotoxicity on SH-SY5Y cells in the concentration range of 200 mu M; the positive drug tert-butylhydroquinone (TBHQ) group has no obvious cytotoxicity to SH-SY5Y cells only in the concentration range of 10 mu M.
2 2 Living HO damaged SH-SY5Y cells (neurodegenerative disease model) by Compounds of formula I and formula II
Sex experiments:
by H 2 O 2 Damaged SH-SY5Y cells are used as model cells of neurodegenerative diseases, and the anti-neurodegenerative activity of the compounds is evaluated by using the CCK-8 method.
The cell viability was assessed by CCK-8 assay, which comprises the steps of:
SH-SY5Y cells or H 2 O 2 (350. Mu.M) injured SH-SY5Y cells were seeded on 96-well plates for 24 hours;
mu.L of 10% (v/v) CCK-8 solution was added and the cells were placed in a dark incubator for 2 hours;
absorbance (OD) values were recorded at 450 nm.
Cell viability was calculated by the following formula: cell viability% = (experimental or model OD-blank OD)/(normal OD-blank OD) ×100%; the results of cell viability were obtained from the mean of the standard deviations (n=3), wherein,
blank group: adding only the culture medium group;
normal group (Normal Control): untreated group;
model group (H) 2 O 2 inst): by 350 mu M H 2 O 2 Inducing a lesion group;
experiment group 1: adding compound 1+350 mu M H with different concentration gradients 2 O 2 A treatment group;
experiment group 2: adding compound 2+350 mu M H with different concentration gradients 2 O 2 A treatment group;
experiment group 3: adding compound 3+350 mu M H with different concentration gradients 2 O 2 A treatment group;
experiment group 4: adding different concentrations of positive medicine tert-butylhydroquinone (TBHQ) +350 mu MH 2 O 2 A treatment group;
wherein, the compounds 1, 2 and 3 are respectively shown in the formulas II-1, I-1 and I-2.
Results: as shown in FIG. 4, compounds 1 to 3 dose-dependently protected SH-SY5Y cells from oxidative damage in the range of 0 to 25. Mu.M at a concentration of 25. Mu.M; the recovery level of cell viability for lesions was higher than that of the positive drug TBHQ (see fig. 5).
In conclusion, the compound with the anti-neurodegenerative change activity can be produced by adopting the mode of co-culturing two strains of the alternaria brassicae and the penicillium, the research methods of changing the culture medium, the culture condition, the mixed culture proportion and the like are changed, the epigenetic regulation is carried out on the silent biosynthesis genes, the capability of synthesizing secondary metabolites of microorganisms is mined as much as possible, and the amplification culture can be carried out on the basis of repeatable and sustainable development of the process, so that the resource waste is avoided.
The compound has neuroprotective activity on cells of a neurodegenerative change disease model, and shows better protective effect than a positive drug TBHQ (tert-butylhydroquinone) at a concentration of 25 mu M.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (9)
1. Compounds having anti-neurodegenerative activity, e.g.
Formula II-1.
2. A formulation for combating neurodegenerative disorders, said formulation comprising a compound of claim 1, and a pharmaceutically acceptable adjuvant.
3. The formulation of claim 2, wherein the pharmaceutically acceptable adjuvant comprises at least one of a diluent, carrier, and excipient.
4. A process for the preparation of a compound as claimed in claim 1, comprising the steps of:
respectively obtaining activated colonies containing alternaria brassicae and penicillium;
inoculating the activated colony to a fermentation medium for fermentation to obtain a fermentation broth; and
extracting and separating the fermentation liquor to obtain the compound;
wherein, the brassica alternaria alternata, latin name Alternaria brassicicola, is from China center for agricultural microbiological culture collection center, deposit number: ACCC 37296;
penicillium graininess, latin name Penicillium granulatum, from China center for collection of marine microorganism strains, accession number: MCCC 3a00475.
5. The method according to claim 4, wherein the activated colony containing Alternaria brassicae and Penicillium brassicae is obtained by inoculating the strain into PDA culture medium and culturing at 28deg.C.
6. The method of claim 4, wherein the fermentation medium comprises 50wt% rice.
7. The method according to claim 4, wherein the step of extracting and separating comprises:
obtaining a crude extract, wherein the crude extract is obtained by extracting the fermentation liquor with ethyl acetate;
obtaining a fine extract, wherein the fine extract is obtained by extracting the crude extract sequentially by petroleum ether, dichloromethane and ethyl acetate; and
the compound is obtained by purifying the refined extract through normal phase silica gel chromatography, reverse phase silica gel chromatography and high performance preparation chromatography, and the compound is the compound of claim 1.
8. The method according to claim 7, wherein the step of obtaining said compound comprises:
obtaining a first component, wherein the first component is obtained by loading the refined extract to a normal phase silica gel column and collecting eluent;
obtaining a second component, wherein the second component is obtained by loading the first component to a reverse phase silica gel column and collecting eluent;
and (3) obtaining a third component, wherein the third component is obtained by loading the second component to a high performance liquid chromatography column and collecting eluent, and comprises a compound shown as II-1.
9. Use of a compound according to claim 1, or a compound prepared by a method according to any one of claims 4 to 8, in the manufacture of a medicament for the treatment of neurodegenerative disorders.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210197180.1A CN114573537B (en) | 2022-03-01 | 2022-03-01 | Compound with anti-neurodegenerative change activity, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210197180.1A CN114573537B (en) | 2022-03-01 | 2022-03-01 | Compound with anti-neurodegenerative change activity, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114573537A CN114573537A (en) | 2022-06-03 |
| CN114573537B true CN114573537B (en) | 2023-06-27 |
Family
ID=81776357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210197180.1A Active CN114573537B (en) | 2022-03-01 | 2022-03-01 | Compound with anti-neurodegenerative change activity, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114573537B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6300371B1 (en) * | 1998-11-05 | 2001-10-09 | Indian Institute Of Chemical Technology | Rameswaralide and rameswaralide derivatives |
-
2022
- 2022-03-01 CN CN202210197180.1A patent/CN114573537B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114573537A (en) | 2022-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107298671B (en) | Selenolonic acid H from penicillium oxalicum and application thereof in preparing medicine for resisting human colon cancer | |
| JPH06505472A (en) | Bridged cyclic ketal derivatives | |
| CN101760496A (en) | Biological dehydrogenation preparation method of steroid drug intermediate | |
| CN107353274B (en) | Selenolonic acid I from penicillium oxalicum and application thereof in preparation of human esophageal cancer resistant medicine | |
| CN107298672B (en) | Application of seclenic acid I derived from penicillium oxalicum in preparation of anti-human colon cancer drugs | |
| CN108640968B (en) | Mixed-source terpenoid and application thereof in preparation of anti-inflammatory drugs | |
| CN107298670B (en) | Application of medicine derived from penicillium oxalicum seclenum ketonic acid H in preparation of anti-human oral epidermoid carcinoma medicines | |
| CN107485607B (en) | Application of seclenic acid H derived from penicillium oxalicum in preparation of human esophageal cancer resistant medicine | |
| CN109134574B (en) | Steroid compound, preparation method and application thereof, and anti-tumor drug | |
| KR101653586B1 (en) | Fructosylated mangiferin and preparation method thereof and use thereof | |
| CN114573537B (en) | Compound with anti-neurodegenerative change activity, preparation method and application thereof | |
| CN109134416B (en) | Application of seclenic acid H derived from penicillium oxalicum in preparation of human cervical cancer drugs | |
| CN109988129A (en) | Compound and its preparing the application in antituberculotic | |
| CN115109023A (en) | Macrolide compound FWYZ52-A, and fermentation strain, fermentation method and application thereof | |
| CN113387788B (en) | Cavity ketone compound and preparation method and application thereof | |
| CN110407797B (en) | Secalonic acid K compound derived from penicillium oxalicum and preparation method thereof | |
| CN111278985B (en) | Novel compound having therapeutic activity against mycobacterium avium complex infection disease and method for producing same | |
| CN109134417B (en) | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human cervical cancer | |
| CN110698537B (en) | Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof | |
| WO2018056470A1 (en) | Epithelial-mesenchymal transition induced cell inhibitor | |
| CN110437314B (en) | Natural Rakicidins compound Rakicidin B1-1 and fermentation extraction method thereof | |
| CN113943350B (en) | Cyclic peptide compound or pharmaceutically acceptable salt thereof, preparation method and application thereof, medicine and application thereof | |
| CN115703788B (en) | Cinnamyl streptomycin and application thereof | |
| CN110698541B (en) | Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof | |
| CN110698466B (en) | Carbon-skin phthalide compound, composition, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |