CN111018802A

CN111018802A - Compound with Parkinson's disease resistance, preparation method and application thereof

Info

Publication number: CN111018802A
Application number: CN201911221903.1A
Authority: CN
Inventors: 胡琳珍; 田书璎; 吴蓉蓉; 胡萍; 张勇慧
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-04-17
Anticipated expiration: 2039-12-03
Also published as: CN111018802B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a compound with an anti-Parkinson's disease effect, a preparation method and an application thereof. The invention cultures and ferments aspergillus ochraceus in deep sea water of pacific ocean, adopts a strategy of a single strain and a plurality of secondary metabolites, and is researched by changing a culture medium and culture conditions, mixing and culturing, particularly adding an enzyme inhibitor and the likeBy means of epigenetic regulation of its biosynthetic gene, new compounds 1 and 2 are separated from metabolic product and MPP is obtained⁺The protection effect of the damaged SH-SY5Y cell-PD model cell apoptosis is an anti-PD activity evaluation index, and the anti-PD activity of the compound is evaluated by using a flow cytometry and thiazole blue cell survival rate detection method. It is found that the compounds 1 and 2 can obviously inhibit MPP⁺Induced SH-SY5Y cell death. The compounds can be expected to be developed into drugs for treating Parkinson's disease and other related neurodegenerative diseases.

Description

Compound with Parkinson's disease resistance, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a compound with an anti-Parkinson's disease effect, a preparation method and an application thereof.

Background

Parkinson's Disease (PD) was first described in 1817 by the British physician James Parkinson as a parkinsonism, one of the most common degenerative changes in the central nervous system, and is clinically manifested by dyskinesias such as myotonia, tremor, and bradykinesia. Epidemiological data indicate that the prevalence rate of PD in older people of 65 or more is about 1.7% in China, and the prevalence rate and incidence rate of PD increase with age. The cure rate for PD patients after treatment does not exceed 2%, the average life span is typically 7 to 14 years, and about 10.3 million people die each year. With the increasing aging degree of the world population, PD, as a representative neurodegenerative disease, has become a main health killer after malignant tumors and cardiovascular and cerebrovascular diseases, and the severity of the symptoms of PD has great influence on the quality of life of patients and families.

The clinical treatment of PD mainly comprises surgical treatment and chemical drug treatment. Surgical treatment may cause severe adverse reactions and a high rate of postoperative recurrence, thereby limiting the widespread use of surgical therapies. Chemical drugs such as levodopa (L-DOPA), amantadine, anticholinergic drugs, etc. have poor efficacy in clinical treatment, or have clinically manifested as worsening of motor function, or unacceptable side effects. Therefore, the screening and the research and development of novel anti-PD chemical drugs have important significance for treating PD.

The prior art has the following disadvantages:

1. at present, the main means for searching the therapeutic medicine from natural products is to extract and separate active ingredients from animal and plant medicines, the period is long, and the resources of animals and plants are easily deficient and even exhausted.

2. The active ingredients of the secondary metabolites of animal and plant medicines in different producing areas and different seasons and the contents thereof are different, so that the process is difficult to reproduce.

3. The clinical existing anti-Parkinson's disease drugs show adverse reactions or side effects of different degrees.

Disclosure of Invention

The invention provides a compound with Parkinson's disease resistance, a preparation method and application thereof, aiming at solving part of problems in the prior art or at least alleviating part of problems in the prior art.

The invention is realized by the compound for resisting Parkinson's disease, the structural formula of the compound is shown as the structure of a compound 1 or a compound 2 in the following formula (I),

the preparation method of the compound with the anti-Parkinson's disease function comprises the following steps: activating ochratoxin in a PDA (potato dextrose agar) culture medium added with a P450 oxidase inhibitor, inoculating the ochratoxin in a rice and water mixed culture medium for fermentation, leaching fermentation liquor with ethanol after fermentation is finished, decompressing and recovering a solvent from an extracting solution to obtain a crude extract, suspending the crude extract with hot water, sequentially extracting with isovolumetric petroleum ether, dichloromethane and ethyl acetate, recovering the solvent from an ethyl acetate extracting part to obtain an extract 1, and recovering the solvent from a dichloromethane extracting part to obtain an extract 2;

carrying out dichloromethane-methanol gradient elution on the extract 1 by normal phase silica gel chromatography to obtain 12 fractions which are sequentially named as Fr.1-Fr.12, carrying out dichloromethane-methanol gradient elution on a component Fr.7 by normal phase silica gel chromatography, and then carrying out methanol elution on the component Fr.7 by a Sephadex LH-20 gel column to obtain a fraction Fr.7.2; fr.7.2 is subjected to high performance liquid chromatography and reversed phase chromatographic column to prepare a compound 1;

mixing the extract 2 with reverse phase silica gel, performing medium pressure reverse phase chromatography, performing gradient elution with methanol-water, detecting by TLC, mixing the same components to obtain 6 fractions named as Fr.1-Fr.6; and repeatedly carrying out normal phase silica gel chromatography, reverse phase silica gel chromatography and Sephadex LH-20 gel column on the component Fr.3, and finally carrying out high performance liquid chromatography and reverse phase chromatography to obtain the compound 2.

Further, when the extract 1 and/or the component Fr.7 are subjected to gradient elution by normal phase silica gel chromatography with dichloromethane-methanol, the range of dichloromethane to methanol is 100:0-0: 100.

When the fraction Fr.7.2 of the extract 1 is treated by a reversed phase chromatographic column, the mobile phase is methanol and water which are 30:70 and 2 ml/min.

Further, when extract 2 was treated by medium pressure reverse phase chromatography, methanol: the range of water is 10:90-0: 100.

Further, when the component Fr.3 of the extract 2 is treated by using a reversed phase chromatographic column, the mobile phase is methanol and water, wherein the ratio of the mobile phase to the mobile phase is 58:42, and the mobile phase is 2 ml/min.

The application of the compound with Parkinson's disease resistance in preparing the medicine for resisting Parkinson's disease.

The application of the compound with Parkinson's disease resistance in preparing the medicine for treating neurodegenerative diseases.

Aspergillus ochraceus (Aspergillus ochraceus) belongs to Aspergillus of the family Tricholomataceae and is a fungus with extremely strong spore-forming capability. Aspergillus ochraceus contains abundant secondary metabolites and takes alkaloid compounds as main metabolites. Modern pharmacological research shows that the alkaloid compound has various pharmacological activities of sedation, depression resistance, spasm resistance, virus resistance, bacteria inhibition, inflammation diminishing and the like, and also has a neuroprotective effect. Under the guidance of the anti-Parkinson's disease activity screening, the invention carries out systematic anti-PD active ingredient research on Aspergillus ochraceus from the deep sea of the Pacific ocean, and separates and identifies 2 new compounds. The structural formula of the compound is shown as a formula (I).

Selection pair MPP⁺Apoptotic of SH-SY5Y cells (PD model cells) of injured human neuroblastoma cellsThe protection effect is an anti-PD activity evaluation index, and the anti-PD activity of the compound is evaluated by using flow cytometry and a thiazole blue detection cell survival rate method (MTT method). The results of the study are as follows: 1. through data analysis of a flow cytometer, the monomer compound does not produce apoptosis effect, MPP, on SH-SY5Y cells⁺The apoptosis number of the treated SH-SY5Y cells is obviously increased, and the MPP can be obviously reduced by the pretreatment of the compound 2⁺The compound 1 has potential MPP inhibition effect on apoptosis of cells⁺Apoptotic effects on cells; 2. through the analysis of data of the MTT method, the compound 2 can inhibit MPP in a dose-dependent manner⁺The induced SH-SY5Y cell death shows that the compound has better anti-Parkinson's disease activity and certain neuroprotective effect. Compound 2 can be expected to develop into drugs against Parkinson's disease and other neurodegenerative diseases.

In summary, the advantages and positive effects of the invention are:

1. the invention adopts a strategy of a single strain and a plurality of secondary metabolites (OSMAC), performs epigenetic regulation on the biosynthesis genes by changing a culture medium and culture conditions and mixed culture, particularly adding enzyme inhibitors and other research means, exploits the capability of microorganisms to synthesize secondary metabolites as far as possible, can infinitely amplify fermentation culture, can realize sustainable development of production process, and cannot cause resource shortage.

2. The invention only needs to recover and subculture the frozen strains, can obtain the target compound through infinite amplification fermentation, and can regulate and control the yield of the compound.

3. Pharmacological activity experiment results show that the

novel compounds

1 and 2 provided by the invention can obviously inhibit MPP⁺The induced SH-SY5Y cell death has anti-Parkinson activity, and can be expected to be developed into drugs for treating Parkinson's disease and other neurodegenerative diseases, such as Alzheimer's disease and the like.

Drawings

FIG. 1 is a single crystal structure of Compound 1;

FIG. 2 is a single crystal structure of Compound 2;

FIG. 3 is a mass spectrometric analysis of Aspergillus ochraceus under PDA medium conditions without addition of CYP450 oxidase inhibitor;

FIG. 4 is a mass spectrometric analysis of Aspergillus ochraceus under PDA medium conditions with addition of the CYP450 oxidase inhibitor furocoumarin 8-Geranyloxypsoralen;

FIG. 5 is the protective effect of

compounds

1 and 2 on apoptosis in a PD model;

FIG. 6 is the combination of

compounds

1 and 2 vs MPP⁺Effect of damaged SH-SY5Y cells-cell survival in PD model.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention provides a compound with Parkinson disease resistance, a preparation method and application thereof. The Aspergillus ochraceus related to the invention is derived from deep seawater of pacific ocean, has the name of Aspergillus ochracea, is shared by China marine microorganism strain preservation management center, and has the following preservation number: MCCC 3A00521, which is available to the public by contacting China center for culture Collection of marine microorganisms.

EXAMPLE 1 preparation of Compounds having anti-Parkinson's disease

1. Fermentation and extraction

Aspergillus ochraceus stored at 4 ℃ is inoculated to potato glucose agar medium (PDA medium) sterilized at high temperature and added with P450 oxidase inhibitor (in the example, CYP450 oxidase inhibitor furocoumarin 8-Geranyloxypyralen is added at 0.25mg/mL) by adopting a strategy of a single strain and multiple secondary metabolites (OSMAC), and the potato glucose agar medium is activated for 7 days in an incubator at 28 ℃ to serve as a seed plate. Mixing 20.0kg rice with water at a ratio of 1:1, sterilizing at 121 deg.C under 15psi for 30 min, cooling to room temperature, inoculating Aspergillus ochraceus strain, fermenting at 25 deg.C under normal pressure and oxygen content in air for 28 days, cold soaking and extracting with 40L 95% ethanol, recovering solvent under reduced pressure to obtain 400g crude extract, suspending with 5L 50 deg.C hot water, and sequentially extracting with equal volume of petroleum ether, dichloromethane and ethyl acetate. Recovering solvent from the extraction part of dichloromethane to obtain 80g of extract; and recovering the solvent from the ethyl acetate extraction part to obtain 40g of extract.

2. Separation of

Subjecting 40g of ethyl acetate extract to normal phase silica gel chromatography (60-80 mesh) with dichloromethane-methanol (100:0-0:100) gradient elution to obtain 12 fractions (Fr.1-Fr.12). And (3) performing normal-phase silica gel column chromatography on the component Fr.7, performing gradient elution by using dichloromethane and methanol in a ratio of 100:0-0:100, and performing Sephadex LH-20 gel column chromatography on the component Fr.7 and the component Fr.2 by using methanol. Fr.7.2 compound 1 was prepared by high performance liquid chromatography using a reverse phase chromatography column (mobile phase: methanol: water: 30:70,2 ml/min).

80g of dichloromethane part extract is evenly stirred by reverse phase silica gel, and then is subjected to medium pressure reverse phase chromatography, and the content of methanol: gradient elution with water 10:90-0:100 followed by TLC detection combined with identical fractions gave 6 fractions a-F (fr.1-fr.6). And repeatedly carrying out normal-phase silica gel chromatography, reverse-phase silica gel chromatography and Sephadex LH-20 gel column on the component Fr.3, and finally carrying out high performance liquid chromatography to prepare the compound 2 by using a reverse-phase chromatographic column (a mobile phase: methanol: water: 58:42,2 ml/min).

EXAMPLE 2 structural characterization of the Compounds

The absolute configuration of

compounds

1 and 2 was obtained by NMR (nuclear magnetic resonance) and X-Ray (X-single crystal diffraction) methods.

1. Nuclear magnetic resonance

Ochracheol a (compound 1): colorless massive crystals with the melting point of 120.6 ℃; HRESIMS [ M + H ]]⁺m/z206.0724 (calculated value C)₁₁H₁₂NO₃,206.0817)；[α]20D+35.2(c 0.61,CH₃OH)；UV(CH₃OH)λ_max(logε)＝274(4.43),208(4.26)nm；IR(KBr)ν_max3402,3390,2938,1620,1505,1446,1384cm^–1；¹H and¹³c NMR data, see Table 1.

Circumstatin N (compound 2): colorless needle crystals; melting point 168.6 deg.C; HRESIMS [ M + Na ]]⁺m/z330.0839 (calculated value C)₁₇H₁₃N₃NaO₃,330.0855)；

(c 0.35,MeOH)；ECD(MeOH)λ(Δε)228(+28.26)；209(-49.82)nm；UV(CH₃OH)λ_max(logε)＝230(4.56),277(4.06)nm；IR(KBr)ν_max3375,3064,1680,1661,1615,1486,1469,1385cm^–1；¹H and¹³c NMR data, see Table 1.

Table 1.¹H(400MHz)and¹³C(100MHz)NMR data of

compounds

1 and 2

^aMeasured in CD₃OD；^bMeasured in DMSO-d₆

2. Diffraction of X-single crystal

The absolute configuration of

compounds

1 and 2 was determined by copper target X-Ray analysis of

compounds

1 and 2, see fig. 1 and 1.

The resulting compounds 1 and 2 were finally determined to have the structural formula (I):

example 3 OSMAC policy

The invention adopts a strategy of a single strain and a plurality of secondary metabolites (OSMAC), and performs epigenetic regulation on a biosynthesis gene by changing a culture medium and culture conditions, mixed culture, particularly research means such as adding an enzyme inhibitor and the like. Finally, it was found that the addition of the enzyme inhibitor had a greater impact on the production of both compounds.

A small amount of Aspergillus ochraceus incubated in a normal PDA medium without CYP450 oxidase inhibitor was dissolved in chromatographic methanol, filtered through a needle-type organic filter, and analyzed by liquid chromatography-mass spectrometry (Finnigan LCQ HPLC/MS) to obtain the spectrum shown in FIG. 3.

A small amount of Aspergillus ochraceus fungus incubated in PDA medium supplemented with CYP450 oxidase inhibitor furocoumarin 8-Geranyloxypsoralen was dissolved in chromatographic methanol, filtered through a needle-type organic filter, and analyzed by liquid chromatography-mass spectrometer (Finnigan LCQ HPLC/MS) to obtain the spectrum shown in FIG. 4.

Mass spectrogram analysis shows that aspergillus ochraceus obtained by adding the medium with CYP450 oxidase inhibition contains more abundant secondary metabolites. The m/z values of 206 and 330 in FIG. 4 are the excimer ion peaks of

compounds

1 and 2, while the m/z peaks of

compounds

1 and 2 are hardly contained in FIG. 3.

EXAMPLE 4 study of the use of Compounds with anti-Parkinson's disease

Selection pair MPP⁺The protection effect of the damaged SH-SY5Y cell-PD model cell apoptosis is an anti-PD activity evaluation index, and the anti-PD activity of the compound is evaluated by using a flow cytometry and thiazole blue detection cell survival rate method (MTT method).

1. Flow cytometry detection of compound protection effect on PD model cell apoptosis

SH-SY5Y cells were inoculated into 100ml flasks and either pre-treated with

Compounds

1 and 2 or MPP alone after attachment to the wall⁺Treatment until the cells have grown to a confluence of about 80% (about 1X 10)⁶One) was digested with pancreatin and the reaction was stopped by adding serum-containing DMEM medium in time. Centrifuging at 800rpm for 5min to collect cells, discarding supernatant, adding culture medium, fully blowing to obtain single cell suspension, and performing operation according to PE-Texas RED-A/FITC kit instructions, and quantifying with flow cytometer. The detailed experimental procedure is as follows:

flow cytometric experimental procedure

Cell culture: SH-SY5Y cells were grown in 10% FBS, 1% double antibody DMEM medium. Culturing the cells at 37 deg.C with 5% CO₂When the cells grow to 80%, the cells are inoculated in a 12-well plate, and induction and administration are carried out after the cells enter a logarithmic growth phase.

Experimental grouping and treatment:

blank group (control): without MPP addition⁺And a medicament;

control (compound 1/2): without addition of MPP⁺Adding only compound 1 or 2;

model set (MPP)⁺): adding 1mM MPP⁺Acting for 24h without adding medicines;

administration set (compound 1 and compound 2): adding 1mM MPP⁺After 24h, the supernatant was discarded and 40 μ M

compound

1 or 2 was added (the present invention also performed at 10 μ M and 20 μ M administration concentrations, but no significant effect was observed, and the administration concentration was 40 μ M, so this example shows the experimental effect at 40 μ M concentration in detail).

Flow-type experiment: cells were digested with 0.25% pancreatin and 2% BSA was added to prevent over-digestion.

(1) Diluting 10-Binding Buffer into 1-Binding Buffer by deionized water;

(2) cell collection: digesting and collecting by using pancreatin without EDTA, centrifuging for 5 minutes at 2000rpm at room temperature, and collecting cells;

(3) and (3) washing cells: resuspend cells once in precooled 1 × PBS (4 ℃), centrifuge for 5 minutes at 2000rpm, discard and wash cells;

(4) 300 μ L of 1 binding buffer suspension cells were added;

(5) annenin V-FITC labeling: add 5 u L Annenin V-FITC mixed, light-proof, room temperature incubation for 15 minutes;

(6) PI marking: 5 mu L of PE-Texas Red A is added for dyeing 5 minutes before the machine is operated;

(7) before loading, 200. mu.L of 1 binding buffer was added.

The results are shown in FIG. 5: through data analysis of a flow cytometer, the

monomeric compounds

1 and 2 do not produce apoptosis effect, MPP, on SH-SY5Y cells⁺The apoptosis number of treated SH-SY5Y cells is obviously increased, and when the administration concentration is 40 mu M, the pretreatment of the compound 2 can obviously reduce the apoptosis effect of MPP + on the cells, and the compound 1 can inhibit the apoptosis effect of MPP + on the cells to a certain extent.

2. MTT method for detecting inhibition rate of compound on PD model cell death

With no addition of MPP⁺Damaged SH-SY5Y cells are used as negative control, and thiazole blue is used for detectionThe cell viability assay (MTT assay) evaluated the anti-PD activity of the compounds.

It was found by preliminary experiments that compounds 1 and 2 both showed significant protection of PD model cells at concentrations up to 10 μ M.

Preparing single cell suspension from cells growing normally in logarithmic growth phase at a cell concentration of 2 × 10⁴And (4) inoculating the cells per ml to a 96-well plate, culturing for 24h, observing the cell state, adhering the cells to the wall overnight, and dividing the cells into a normal control group, a model group and an administration group, wherein each group is provided with 6 multiple wells. The normal group was added with fresh medium and the model group was added with MPP to a final concentration of 1mM⁺The administration group was previously added with 1.0. mu.M, 1.5. mu.M and 2.0. mu.M of

Compound

1 or 2. Adding MPP with final concentration of 1mM after 1h⁺Placing at 37 ℃ and 5% CO₂After the culture is continued for 24h in the incubator, the culture solution is carefully removed, then 100 mul of DMSO is added, the culture plate is shaken for 10min by applying a set program of a microplate reader, further, the crystals are completely dissolved, the OD value at the position of 492nm of wavelength is detected, and the cell survival rate is calculated. The experiment was repeated 3 times and the mean value was taken for analysis.

The results are shown in FIG. 6: through the data analysis of the MTT method, the MPP pair of the

compounds

1 and 2 is found⁺The damaged SH-SY5Y cell-PD model cell has a certain protective effect and a certain dose-effect relationship, and the cell survival rate is increased along with the increase of the concentration of the compound. The compound 2 can obviously inhibit MPP when the concentration is 2.0 mu M⁺The induced cell death shows that the compound has better anti-Parkinson's disease activity and certain neuroprotective effect.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The compound with the Parkinson disease resistance is characterized in that the structural formula of the compound is shown as the structure of a compound 1 or a compound 2 in the following formula (I),

2. the method of claim 1, wherein the method of making compound 1 comprises the steps of: activating ochratoxin in a PDA (potato dextrose agar) culture medium added with a P450 oxidase inhibitor, inoculating the ochratoxin in a rice and water mixed culture medium for fermentation, leaching fermentation liquor with ethanol after fermentation is finished, decompressing and recovering a solvent from an extracting solution to obtain a crude extract, suspending the crude extract with hot water, sequentially extracting with isovolumetric petroleum ether, dichloromethane and ethyl acetate, recovering the solvent from an ethyl acetate extracting part to obtain an extract 1, and recovering the solvent from a dichloromethane extracting part to obtain an extract 2;

carrying out dichloromethane-methanol gradient elution on the extract 1 by normal phase silica gel chromatography to obtain 12 fractions which are sequentially named as Fr.1-Fr.12, carrying out dichloromethane-methanol gradient elution on a component Fr.7 by normal phase silica gel chromatography, and then carrying out methanol elution on the component Fr.7 by a SephadexLH-20 gel column to obtain a fraction Fr.7.2; fr.7.2 by high performance liquid chromatography and reversed phase chromatographic column to obtain compound 1.

3. The method of claim 1, wherein the method of making compound 2 comprises the steps of: activating ochratoxin in a PDA (potato dextrose agar) culture medium added with a P450 oxidase inhibitor, inoculating the ochratoxin in a rice and water mixed culture medium for fermentation, leaching fermentation liquor with ethanol after fermentation is finished, decompressing and recovering a solvent from an extracting solution to obtain a crude extract, suspending the crude extract with hot water, sequentially extracting with isovolumetric petroleum ether, dichloromethane and ethyl acetate, recovering the solvent from an ethyl acetate extracting part to obtain an extract 1, and recovering the solvent from a dichloromethane extracting part to obtain an extract 2;

4. The process for the preparation of compounds having anti-parkinson's disease properties according to claim 2, characterized in that: and when the extract 1 and/or the component Fr.7 are subjected to gradient elution by normal phase silica gel chromatography with dichloromethane-methanol, the range of the dichloromethane to the methanol is 100:0-0: 100.

5. The process for the preparation of compounds having anti-parkinson's disease properties according to claim 2, characterized in that: when the fraction Fr.7.2 of the extract 1 is treated by a reversed phase chromatographic column, the mobile phase is methanol and water with the ratio of 30:70 and the concentration is 2 ml/min.

6. The method for preparing a compound having an anti-parkinson's disease property according to claim 3, wherein: when extract 2 was treated with medium pressure reverse phase chromatography, methanol: the range of water is 10:90-0: 100.

7. The method for preparing a compound having an anti-parkinson's disease property according to claim 3, wherein: when the component Fr.3 of the extract 2 is treated by a reversed phase chromatographic column, the mobile phase is methanol and water, 58:42,2 ml/min.

8. Use of the compound having an anti-parkinson's disease property according to claim 1 for the preparation of an anti-parkinson's disease medicament.

9. Use of a compound having an anti-parkinson's disease property according to claim 1 for the preparation of a medicament for the treatment of neurodegenerative disorders.