CN102309528B - Preparation method of active substance capable of resisting neurodegenerative disease - Google Patents
Preparation method of active substance capable of resisting neurodegenerative disease Download PDFInfo
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Abstract
The invention relates to the technical field of natural products and particularly relates to a method for extracting an active substance capable of resisting the neurodegenerative disease from mangrove salt-resistant endophytic fungi. The method comprises the following steps: fermenting and culturing the mangrove salt-resistant endophytic fungi, filtering and collecting thalli; performing vacuum drying on the thalli at a low temperature; 3) leaching the thalli with methanol; 4) steaming methanol leaching liquid at a low temperature to remove methanol and then performing liquid-liquid partition with water and normal butanol; and 5) concentrating the water-phase part, then performing Diaion-20 resin column chromatography, and performing gradient elution by utilizing water and methanol as a flow phase, thus obtaining a fluid with obvious degenerative disease resisting activity. According to the invention, after an Abeta42 aggregation model and a Poly Q aggregation model are utilized for the screening and research on the neurodegenerative disease resisting activity, the active substance provided by the invention is proved to have strong effects of gathering and inhibiting the key protein of the neurodegenerative disease.
Description
Technical field
The present invention relates to the natural product technical field, relate in particular to a kind of the extraction and have the method for remarkable anti-nerve degenerative diseases active substance from hide Phomopsis.
Background technology
Nerve degenerative diseases comprise A Ercicaimo sick (Alzheimer ' s disease, AD), Parkinson's disease (Parkinson ' s disease, PD), Huntington Chorea (Huntington ' s disease, HD) etc., be a kind of frequently-occurring disease and common disease clinically, cardinal symptom comprises carrying out property memory and cognition dysfunction, dyskinesia etc.In case morbidity causes great spirit and economical load can for patient, family and society.Along with the aging of human society age structure, the nerve degenerative diseases sickness rate raises increasingly, has become the third-largest worldwide health problem that is only second to cardiovascular and cerebrovascular diseases and tumour.
People have carried out large quantity research to the pathogeny of nerve degenerative diseases in recent years, also some different hypothesis have been proposed its pathogeny, as entanglement and gathering, the oxidative stress of key protein, the biological metal ionic homeostasis is unbalance, mitochondrial function exhaustion, inherited genetic factors etc.Yet the pathogenic factor of nerve degenerative diseases and mechanism are still not very clear at present.Existing result of study shows, the aggregate and precipitate of the relevant key protein of nerve degenerative diseases has important regulative in pathogenic process, as namely directly related with the cytotoxicity that gathering, precipitation and the oligomer of the key protein such as A β (beta-amyloid) produce in the morbidity of AD, the overexpression of aβ protein and gathering, precipitation can cause directly that mitochondrial disorders, oxidative stress, nerve synapse transmission interruption, aixs cylinder transportation are interrupted, the impaired multiple AD related pathologies such as break of film integrality changes, and the morbidity of HD is directly related with the gathering of Poly Q etc.Therefore, the aggregate and precipitate of nerve degenerative diseases associated protein has become one of research nerve degenerative diseases pathogeny and the most important target of anti-nerve degenerative diseases medicine.
Although the nerve degenerative diseases associated protein forms insoluble fibre by the folding crosslinked gathering of β sheet, the death of neurocyte finally occurs and cause with block high polymer form.But result of study proves recently, and the soluble proteins oligomer that produces in forming the insoluble fibre process is only its neurovirulent material of real embodiment.Therefore, compare with the aggregation inhibitor of research fiber or block high polymer, how inquiring into, the aggregation inhibitor of screening study nerve degenerative diseases associated protein oligomer has prior theory significance and value for anti-nerve degenerative diseases study of active components.
With slowly deeply comparing of nerve degenerative diseases curative mechanism research, the correlative study of anti-nerve degenerative diseases medicine obviously lags behind, and there is no clinically up to now etiotropic specific treatment medicine.Existing medicine only has certain auxiliaring effect to the state of an illness of nerve degenerative diseases, can delay the deterioration of the state of an illness to a certain extent, but all can not reverse disease, be example as anti-AD medicine, existing acetylcholinesterase depressant (IChEIs) is as tacrine, donepezil, huperzine, ENA-713, fuperdinA etc. and for the clinical medicines such as uncompetitive nmda receptor antagonist hydrochloric acid memantine (memantine), certain adjuvant treatment effect was only arranged all in recent years, also can cause the vagusstoff related reactions in a lot of situations.Therefore, that research is found is new, the anti-nerve degenerative diseases medicine resource of special habitats and find that therefrom the unique novel active composition of novel structure, effect is very important for the research of anti-nerve degenerative diseases lead drug, is one of core means that solve the nerve degenerative diseases new drug development.
Mangrove forest is a kind of very special ecological zone between marine ecology and land ecology.The impact that is subjected to mangrove forest height salt dutyization, the anoxic of soil, high optical radiation and periodic seawater to soak unique growing environments such as flooding, the very abundant mangrove microorganisms resource that has again characteristic has become one of unique Ocean Medicinal resource.After separation obtained producing the endogenetic fungus Taxamyces andrenae of novel active from Pacific yew (yewtree) from 1993, the research of mangrove plant endogenetic fungus active substance had caused people's very big interest.At present, both at home and abroad in mangrove endogenetic fungus secondary metabolite isolation identification unique, the novel compounds of a plurality of structures, larger application prospect is arranged in tumour, virus, the major disease such as antibiotic aspect preventing and treating.Wherein the sporothrins A of isolation identification and Xylaria sp. meta-bolites Xyloketal A-D have stronger restraining effect to acetylcholinesterase from Kandelia candel bark endogenetic fungus thalline.This just further proves: mangrove endogenetic fungus and metabolite thereof have great exploitation value as the resource of anti-nerve degenerative diseases new drug research.
Summary of the invention
Main purpose of the present invention is to provide a kind of and extracts preparation have the preparation method of remarkable anti-nerve degenerative diseases active substance from hide Phomopsis, extracts the preparation obtained component and has stronger nerve degenerative diseases key protein and assemble and suppress active.
Mangrove salt tolerance endogenetic fungus of the present invention Institute of Microorganism, Academia Sinica is accredited as: Phomopsis occulta, hide Phomopsis, it is preserved in Wuhan University's preservation center, Luojiashan, Wuchang, Wuhan City, Hubei Province on March 2nd, 2011, be Chinese Typical Representative culture collection center (CCTCC), deposit number is CCTCC M2011044.This laboratory is to its called after SN3-2.
For achieving the above object, the present invention adopts following technical scheme:
A kind of preparation method of anti-nerve degenerative diseases active substance comprises the following steps:
1) hide Phomopsis and ferment in the basic culture solution that contains 0-3M NaCl, filter, collect thalline;
2) thalline low temperature, vacuum-drying;
3) thalline leaches with methyl alcohol;
4) methyl alcohol leaching liquid low temperature steams except water after methyl alcohol and propyl carbinol distribution extraction;
5) aqueous portion concentrated after with Diaion-20 resin column chromatography, take water and methyl alcohol as moving phase, adopt gradient elution, with the moving phase wash-out of 1%-60% methanol content, the flow point that obtains having anti-nerve degenerative diseases activity.
Described step 2) be specially: the thalline cryodrying 24 hours of reducing pressure in 40 ℃ of constant-temperature vacuum loft drier.
Described step 3) is specially: after dry, thalline soaks with methyl alcohol, and ultrasonic 15 minutes, flooded 3 days, repeat 3 times, filter united extraction liquid, 45 ℃ remove methyl alcohol under reduced pressure and get the thallus extract aqueous solution.
Described step 4) is specially: add the pure water of two volumes in the thallus extract aqueous solution, then the propyl carbinol with 4/3 cumulative volume extracts, and repeats 4 times, propyl carbinol is partly washed 1 time, merge each aqueous portion, 45 ℃ are evaporated to 500 milliliters of cumulative volumes, get the thalline water soluble extract.
The second purpose of the present invention is to provide a kind of anti-nerve degenerative diseases active substance, and it is prepared from by aforesaid method.
Utilize the method for the invention preparation anti-nerve degenerative diseases active substance, carry out the nerve degenerative diseases key protein with external A β 42 Aggregation Model, Poly Q Aggregation Model and assemble inhibition screening active ingredients and research, this active substance has very strong gathering restraining effect.
Utilize the effective constituent of the anti-nerve degenerative diseases active substance of the method for the invention preparation to have water-soluble preferably, all more stable under the aqueous solution, organic solution, neutrallty condition, weak acid alkali condition, still keep the activity more than 95% after 70 ℃ of processing 120min.
Mangrove salt tolerance endogenetic fungus Phomopsis occulta of the present invention has stronger anti-nerve degenerative diseases, contain in a large number the anti-gathering active component take the gathering of nerve degenerative diseases key protein A β 42, Poly Q etc. as target spot in its secondary metabolite, possess the great potential of therefrom finding novel anti-nerve degenerative diseases lead drug, will provide support for the research of novel anti-nerve degenerative diseases medicine.The research of the method for the invention and product antagonism nerve degenerative diseases specific medicament thereof and the deep development of China's South Sea mangrove resource have very important meaning and value.
Description of drawings
Fig. 1 is the external A β 42 Aggregation Model results of study of active substance of the present invention.
Fig. 2 is the external Poly Q of active substance of the present invention Aggregation Model result of study.
Embodiment
Integral Thought of the present invention comprise following some: 1) hide the Phomopsis fermentation; 2) extract anti-nerve degenerative diseases active substance from the fermentation thalline; 3) the detection of active material is assembled the activity that suppresses to the nerve degenerative diseases key protein; Above each point is all independent embodiment, is described in further details below with reference to specific embodiment.
(1) substratum and preparation:
Basic culture solution: peptone (peptone) 0.3%, ammonium sulfate 0.2%, yeast extract paste 0.05%, potassium primary phosphate 0.4%, calcium chloride 0.03%, magnesium sulfate heptahydrate 0.03%, glucose 10g/L adds respectively the different NaCl that measure, and makes its NaCl content be respectively 0-3M.0.1MPa, 121 ℃ of sterilization 30min.
(2) fermentation:
Mangrove salt tolerance endogenetic fungus SN3-2 of the present invention be transferred to respectively contain 0,1,2, in the fungi basic culture solution of 3MNaCl, each 50L coerces cultivation, 25 ℃ of standing for fermentation 40 days obtain fermented liquid.
The anti-nerve degenerative diseases active substance of embodiment 2 hiding Phomopsis of the present invention preparation method
(1) fermented liquid that obtains of embodiment 1 with 9 layers of gauze decompress filter, is collected thalline;
(2) the thalline cryodrying 24 hours of reducing pressure in 40 ℃ of constant-temperature vacuum loft drier;
(3) dry rear thalline soaks with 3L methyl alcohol, and ultrasonic 15 minutes, flooded 3 days, repeat 3 times, filter united extraction liquid, 45 ℃ of decompressions are divided exactly methyl alcohol and are got the thallus extract aqueous solution;
(4) add the pure water of two volumes in the thallus extract aqueous solution, then the propyl carbinol with 4/3 cumulative volume extracts, and repeats 4 times, propyl carbinol is partly washed 1 time, merge each aqueous portion, 45 ℃ are evaporated to 500 milliliters of cumulative volumes, get the thalline water soluble extract;
(5) filling Diaion-20 resin chromatography column, column volume is 300 milliliters, with the thalline water soluble extract with the speed loading of 2 times of column volumes per hour, then begin the column chromatography wash-out take water and methyl alcohol as mobile phase composition, mobile phase composition is that 0% methyl alcohol-60% methyl alcohol gained flow point has higher nerve degenerative diseases key protein and assembles and suppress active.
The external A β 42 Aggregation Model screening studies of the anti-nerve degenerative diseases activity of embodiment 3 hiding Phomopsis of the present invention:
ThT analyze to use synthetic A β 42 polypeptide, 37 ℃ standing 2 hours, add final concentration 100 each samples of μ g/mL, with the fluorescence intensity of fluorescence microplate reader test reaction system, excitation wavelength is 444nm, emission wavelength is 485nm.With the negative contrast of DMSO, the positive contrast of EGCG can judge that according to the difference of system fluorescence intensity component is active to the inhibition that A-beta assembles.Experimental result is referring to Fig. 1.The experimental result explanation: a plurality of sample relative intensity of fluorescence are lower, A-beta42 assembled have remarkable restraining effect, and the sample segment restraining effect is better than positive control sample EGCG or approaches with it.
The external Poly Q Aggregation Model screening study of the anti-nerve degenerative diseases activity of embodiment 4 hiding Phomopsis of the present invention:
ThT analyze to use purifying Poly Q albumen, 37 ℃ standing 2 hours, add final concentration 100 each samples of μ g/mL, with the fluorescence intensity of fluorescence microplate reader test reaction system, excitation wavelength is 444nm, emission wavelength is 485nm.With the negative contrast of DMSO, the positive contrast of EGCG can judge that according to the difference of system fluorescence intensity component is active to the inhibition that Poly Q assembles.Experimental result is referring to Fig. 2.The experimental result explanation: a plurality of sample relative intensity of fluorescence are lower, Poly Q assembled have remarkable restraining effect, and sample segment restraining effect and positive control sample EGCG approach.
Claims (6)
1. the preparation method of an anti-nerve degenerative diseases active substance comprises the following steps:
1) hide Phomopsis and ferment in the basic culture solution that contains 0-3M NaCl, filter, collect thalline;
2) thalline low temperature, vacuum-drying;
3) thalline leaches with methyl alcohol;
4) methyl alcohol leaching liquid low temperature steams except water after methyl alcohol and propyl carbinol distribution extraction;
5) aqueous portion concentrated after with Diaion-20 resin column chromatography, take water and methyl alcohol as moving phase, adopt gradient elution, with the moving phase wash-out of 1%-60% methanol content, the flow point that obtains having anti-nerve degenerative diseases activity.
2. the preparation method of a kind of anti-nerve degenerative diseases active substance according to claim 1, it is characterized in that: the deposit number of described hiding Phomopsis is CCTCC M2011044.
3. the preparation method of a kind of anti-nerve degenerative diseases active substance according to claim 1 and 2, is characterized in that, described step 2) be specially: the thalline cryodrying 24 hours of reducing pressure in 40 ℃ of constant-temperature vacuum loft drier.
4. the preparation method of a kind of anti-nerve degenerative diseases active substance according to claim 1 and 2, it is characterized in that, described step 3) is specially: after dry, thalline soaks with methyl alcohol, ultrasonic 15 minutes, flooded 3 days, repeat 3 times, filter united extraction liquid, 45 ℃ remove methyl alcohol under reduced pressure and get the thallus extract aqueous solution.
5. the preparation method of a kind of anti-nerve degenerative diseases active substance according to claim 1 and 2, it is characterized in that, described step 4) is specially: the pure water that adds two volumes in the thallus extract aqueous solution, then the propyl carbinol with 4/3 cumulative volume extracts, repeat 4 times, propyl carbinol is partly washed 1 time, merges each aqueous portion, 45 ℃ are evaporated to 500 milliliters of cumulative volumes, get the thalline water soluble extract.
6. the anti-nerve degenerative diseases active substance of the described method of claim 1 or 2 preparation.
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