CN108042576A - A kind of antiparkinsonism drug preparation method and applications based on brain-gut axis - Google Patents
A kind of antiparkinsonism drug preparation method and applications based on brain-gut axis Download PDFInfo
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Abstract
The present invention relates to the purposes of Clostridium butylicum, and in particular to application of the Clostridium butylicum preparation in the drug for the treatment of Parkinson's disease belongs to field of biological pharmacy.Intestines brain axis is the two-way exchange system between enteron aisle and brain, is made of neuroendocrine, immune, vagus nerve approach, probiotics can adjust intestinal flora, and influence neurobehavioral by regulating and controlling intestines brain axis.The present invention has developed a kind of good effect, the drug for the anti-Parkinson's disease having no toxic side effect, and is proved through animal experiment study, and bacillus amylobacter preparation can play the effect of anti-Parkinson's disease, has very strong practical value.
Description
Technical field
The present invention relates to the purposes of Clostridium butylicum, and in particular to Clostridium butylicum preparation belongs in the application of anti-Parkinson's disease
Field of biological pharmacy.
Background technology
Parkinson's disease is a kind of serious central nervous system degenerative disease, and incidence is high, difficulty is cured, in often causing
The dyskinesia of the elderly causes to seriously affect to patient health and quality of life.Parkinson's disease (PD) is after Alzheimer
Second largest chronic CNS degenerative disease after disease, with static tremor, myotonia, bradykinesia and Parkinson
Posture abnormal gait is main clinical characteristics.For the onset peak period of Parkinson's disease about at 60 years old, male patient is significantly more than female
Property patient, China's over-65s people's illness rate is about 1.7%.Part PD patient is with cerebral cortex, dorsal nucleus of vagus nerve, locus coeruleus
The degeneration necrosis of the neurons such as core, it may appear that sensory function symptom (such as dysosmia), dementia, cognitive impairment and autonomous god
Through non-motor symptoms such as functional disturbances.In recent years the study found that the non-motor symptoms of Parkinsonian are to the shadow of quality of life
It rings and is even more than motor symptoms.With the quickening of China human mortality aging process, the incidence of Parkinson's disease increasingly increases, morbidity
Number increases year by year, and the Parkinsonian in China is more than 2,000,000 at present.Therefore, Parkinson's disease more and more causes social each
The concern on boundary.
The pathogenesis of Parkinson's disease is complicated, and at present, the pathogenesis on Parkinson's disease mainly has Ubiquitin-proteasome
System theory, excitatory neuron poison theory, oxidative stress, inflammatory reaction, Apoptosis, mitochondria dysfunction, science of heredity because
Element etc..For these pathogenesis, clinically there is a corresponding medicine, application it is more universal include levodopa class system
Agent, monoamine oxidase B (MAO-B) inhibitor, DA receptor stimulating agents, CO2 laser weld preparation etc., but these drug therapy targets
Point is single, and generally existing side effect.For example, almost more than 50% patient is concurrent with there is movement after plan dopaminergic agent
Disease (motor fluctuations or unusual fluctuation disease), these motor complications can aggravate Parkinsonian's state of an illness, seriously affect minimal invasive treatment's matter
Amount, while also increase treatment difficulty.This exactly provides new focus and power for us to PD Therapy studies.
In recent years, the research in relation to pathogenesis of Parkinson disease has focused largely on neurotransmitter, cell factor etc., very
It is related to microorganism field less, but clinically intestinal flora and Parkinson's disease is prompted there are close ties.Research shows alpha-synapse core egg
White mutation or overexpression are one of typical pathological features of Parkinson's disease, and zoopery shows the variation of intestinal microbiota
The inflammatory reaction of enteron aisle can be caused, the false folding of α-SYN may be triggered.The continuous of pathogenesis of Parkinson's disease was studied
Cheng Zhong has found the close relation of intestinal flora and Parkinson's disease.Although Parkinson's disease is a typical dyskinetic disorder,
But also include many non-motor symptoms, such as cognitive impairment, sleep-disorder, mental symptom and most common gastrointestinal dysfunction.Greatly
The epidemiological study of amount finds that many Parkinsonians once endured the puzzlement of enterogastric diseases to the fullest extent before being diagnosed.Pa
The gloomy patient of gold is also present with chronic inflammation infiltration, and proinflammatory disease/anti-inflammatory cytokine ratio rises, and probiotics has well
Anti-inflammatory effect.These discoveries all imply Parkinson's disease and are closely connected with probiotics presence.Some researches show that enterobacteriaceaes
The neurobehaviorals class diseases such as the variation of group structure and autism, chronic fatigue syndrome, Parkinson's disease and anxiety are related, this prompting
We link up enteron aisle and the important pivot brain-gut axis of brain tissue's functional activity plays an important role, with intestinal flora structure
Into intestinal flora-intestines-brain axis, the activity of central nervous system is influenced.
So far, the pathogenesis of Parkinson's disease is not yet clear and definite, in recent years, as people are to intestinal flora regulating and controlling effect
Understanding constantly deepen, it is found that intestinal flora and the nervous system disease are in close relations, intestinal flora can pass through intestines-brain axis influence
Nervous function and neurobehavioral.Numerous studies are found：Probiotic supplemented can improve and treat Parkinson's disease, irritability brain
The psychotic mental illnesses such as damage, and it is increasingly becoming an emerging treatment means.Using intestinal flora as the probiotics of target spot
Intervention is likely to become following improvement and treatment mental illness safely and effectively one for the treatment of means.
Brain-gut axis is the embodiment contacted between nervous system and gastrointestinal tract.More and more people begin to focus on enterogastric diseases
With the relation of neurogenic disease, and attempt by intervening intestinal microecology system enhancement brain diseases.The study found that intestinal flora
Change betide in Parkinson's disease premorbid and the course of disease, the disturbances in patients with Parkinson disease more than 80% is accompanied by different degrees of stomach and intestine work(
It can be disorderly.Intestinal bacilli illness is not only possible to trigger intestines-cerebral disease (such as irritable bowel syndrome, inflammatory bowel disease and liver property brain
Disease), and Central nervous systemic disease (such as multiple sclerosis, alzheimer's disease and self-closing disease) also generates huge shadow
It rings.Studies have found that the probiotics such as Bifidobacterium, Bacillus acidi lactici can improve parkinsonian symptoms.Therefore, using enteron aisle as controlling
It may be a feasible way to treat target treatment Parkinson's disease.Clostridium butylicum (Clostridium butyricum) is to be present in
Probiotics in human body intestinal canal has and maintains or recover enteron aisle dominant microflora, adjusts the function of human body intestinal canal microecological balance.In
State patent CN105012349A discloses a kind of probiotics preparation for the treatment of of vascular dementia and preparation method thereof, is related to butyric acid shuttle
The new medicine use of bacterium treatment of vascular dementia, vascular dementia is dull-witted mainly caused by ischemic lesions, with spirit
Disease pathogenesis of Parkinson disease has completely different difference, and Parkinson's disease is to fall to principal mode with DOPAMINE CONTENT IN RABBIT.At present
Have no report data of the clostridium butyricum on treatment of Parkinson disease.
The content of the invention
The defects of it is an object of the invention to overcome the prior art and deficiency, using Clostridium butylicum preparation in Parkinson's disease
On amelioration of disease and treatment, and then develop the drug for the treatment of Parkinson's disease.
The Clostridium butylicum preparation of a kind of anti-Parkinson's disease, it is characterised in that the preparation method of Clostridium butylicum preparation is as follows：
(1) prepared by seed liquor：Picking single bacterium colony Clostridium butylicum (Clostridium butyricum) WZ001, deposit number
For CGMCC No.8808, it is inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel for 24 hours, obtains seed liquor；
(2) ferment：Containing with weight hundred after step (1) seed liquor has been sterilized by the inoculum concentration access of 1% weight ratio
Divide the peptone 1-5% than meter, yeast extract 0.5-5%, glucose 1-10%, oligofructose 0.5-2%, ammonium sulfate
0.1%th, potassium dihydrogen phosphate 0.05-0.3%, dipotassium hydrogen phosphate 0.05-0.3%, manganese sulfate 0.02%, magnesium sulfate 0.02% and chlorine
Change calcium 0.002%, remaining is in the fluid nutrient medium of deionized water, and anaerobic fermentations -48h for 24 hours is stood through 35 DEG C -37 DEG C；
(3) thalline is collected：Clostridium butylicum (Clostridium butyricum) WZ001 that step (2) culture obtains is sent out
Zymotic fluid, 3000rpm centrifuge 5~10min, remove supernatant, obtain bacterium mud, and 2~3 washings of deionized water are made bacterium and mix
Suspension centrifuges 5~10min with 3000rpm again, removes supernatant fluid and obtain sediment；
(4) bacterium broken wall：By the sediment that step (3) obtains by 1: 5~1: 10 plus deionized water, under 150MPa pressure
Clasmatosis obtains breaking-wall cell product；
(5) degreasing is eluted：The sediment that will be obtained in step (4) carries out dissolving elution repeatedly using ethyl alcohol, and 10,
000rpm centrifuges 15~20min, recycles sediment, and washing steps are 2~3 times, then with the washing of deionized water 3~5 times,
3000rpm centrifuges 5~10min, obtains sediment；
(6) finished product：The sediment obtained in step (5) is freeze-dried under vacuum negative pressure condition, you can obtain purity
Reach more than 95% dried powder to get to Clostridium butylicum preparation.
Clostridium butylicum (Clostridium butyricum) WZ001 of the present invention, on 01 22nd, 2014 are deposited in
State's Microbiological Culture Collection administration committee common micro-organisms center, is referred to as CGMCC (addresses：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is Clostridium butylicum
(Clostridium butyricum) WZ001, deposit number are CGMCC No.8808.
Since present invention firstly discloses preparing answering for treatment anti-parkinson drug by medicament active composition of butyric acid shuttle
With, therefore, medicament is made with auxiliary material combination, as long as the medicament belongs to the protection model of the present invention for treating Parkinson's disease
It encloses.
Advantages of the present invention and effect：
The invention firstly discloses Clostridium butylicums to treat the purposes of Parkinson's disease, and the present invention is with good effect, nontoxic secondary work
The function of anti-Parkinson's disease.
Specific embodiment
Embodiment 1
The preparation method of the Clostridium butylicum preparation is as follows：
(1) prepared by seed liquor：Picking single bacterium colony Clostridium butylicum (Clostridium butyricum) WZ001, deposit number
For CGMCC No.8808, it is inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel for 24 hours, obtains seed liquor；
(2) ferment：Containing with weight hundred after step (1) seed liquor has been sterilized by the inoculum concentration access of 1% weight ratio
Divide the peptone 4% than meter, yeast extract 2.5%, glucose 10%, oligofructose 1%, ammonium sulfate 0.1%, biphosphate
Potassium 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate 0.02%, magnesium sulfate 0.02% and calcium chloride 0.002%, remaining is deionization
In the fluid nutrient medium of water, anaerobic fermentation is stood for 24 hours through 37 DEG C；
(3) thalline is collected：Clostridium butylicum (Clostridium butyricum) WZ001 that step (2) culture obtains is sent out
Zymotic fluid, 3000rpm centrifugation 10min, removes supernatant, obtains bacterium mud, and bacterial suspension is made in 3 washings of deionized water, then
It is secondary to centrifuge 10min with 3000rpm, remove supernatant fluid and obtain sediment；
(4) bacterium broken wall：By the sediment that step (3) obtains by 1: 10 plus deionized water, the cell under 150MPa pressure
It is broken, obtain breaking-wall cell product；
(5) degreasing is eluted：The sediment that will be obtained in step (4) carries out dissolving elution repeatedly using ethyl alcohol, and 10,
000rpm centrifuges 20min, recycles sediment, and washing steps are 3 times, then with the washing of deionized water 5 times, 3000rpm centrifugations
10min obtains sediment；
(6) finished product is freezed：The sediment obtained in step (5) is freeze-dried under vacuum negative pressure condition, you can obtain
Purity reach more than 95% dried powder to get to Clostridium butylicum (Clostridium butyricum) WZ001 activity into
Point.
Clostridium butylicum (Clostridium butyricum) WZ001 of the present invention, on 01 22nd, 2014 are deposited in
State's Microbiological Culture Collection administration committee common micro-organisms center, is referred to as CGMCC (addresses：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is Clostridium butylicum
(Clostridium butyricum) WZ001, deposit number are CGMCC No.8808.
Embodiment 3
First, materials and methods
1. experimental animal and grouping
Male C57BL/B6 mouse, 20 ± 2g of weight purchased from Shanghai Slac Experimental Animal Co., Ltd., are randomly divided into three
Group：Normal group；Parkinson disease model group；1 bacillus amylobacter preparation processing group of embodiment.
2. prepared by the animal model of Parkinson's disease
Using intraperitoneal injection 1- methyl 4-phenyls -1,2,3,6- tetrahydropyridines (MPTP) method prepares parkinsonian mouse
Model.3mg/ml solution is configured to physiological saline solution MPTP, at Parkinson disease model group, 1 bacillus amylobacter preparation of embodiment
MPTP is injected intraperitoneally with 30mg/kg in reason group, one time a day, continuously injects 7d, 1 bacillus amylobacter preparation group processing group mouse of embodiment
8d comes into effect 1 bacillus amylobacter preparation group continuous gavage 14d of example, Normal group intraperitoneal injection normal saline.
3. behaviouristics detects
The clay bead of a diameter of 2.5cm of 3.1 Grasping clubglass tests is fixed on the rod top of a root long 50cm × 1cm, to be measured
Mouse is put on bead, is measured mouse and is got off from bead the required time.Such as mouse midway stopping or knot of reversely creeping
Fruit is disregarded, and retests.Measurement 2~3 times daily of every mouse, it is shortest once as effectively using the used time of continuously creeping.
3.2 suspension experiments are bundled with rubber band at the about 1cm of mousetail end, and rubber band is fixed in hanging rope, is made
Mouse is liftoff 20cm.Every mouse experiment time is 6min, and the suspension of 1min mouse adapts to, and records the dead time of 5min mouse.
Mouse to be measured is put into the beaker for filling 1000ml water by 3.3 forced swim tests, and total testing time is 6min,
1min is adapted to, the dead time of record 5min mouse floatings.Motionless status and appearance still swims in the water surface or only permits for mouse
Perhaps there are some rotations on its head.
4.Nissl is dyed and Immunohistochemical detection
After Behavior test, mouse thoracic cavity is opened, PBS and 4% paraformaldehyde is perfused, until small in exposure heart successively
Mouse is stiff, takes out brain tissue and 72h is fixed into paraformaldehyde.Mice brain tissues make pathology and cut by embedding, section, dyeing
Piece, high power Microscopic observation are simultaneously taken pictures.Immunohistochemical staining is operated according to antibody specification, primary antibody for rabbit-anti TH and
Occludin, secondary antibody are rabbit two step method kit.
5.Western Blot
Primary antibody is that rabbit-anti Occludin, β-actin are internal reference；Secondary antibody marks goat anti-rabbit igg for HRP.
2nd, result and analysis
1. Grasping clubglass test
The Parkinson's disease group pole-climbing time grows (P ＜ 0.05) compared with Normal group, and Parkinson disease mice is shown accordingly
Symptom, and the pole-climbing time of 1 bacillus amylobacter preparation processing group mouse of embodiment is significantly lower than Parkinson's disease group (P ＜ 0.05).
2. forced swim test and suspension are tested
In forced swim test, the flotation time more normal control group mice of Parkinson disease mice is obviously prolonged (P
＜ 0.01)；In suspension is tested, Parkinson disease model group mouse does not drop the time less than Normal group, with Parkinson's disease mould
Type group compares, and the mouse flotation time of 1 bacillus amylobacter preparation group of embodiment is obviously shortened (P ＜ 0.01), and suspension is not dropped the time
It is obviously prolonged.
3.Nissl is dyed
Tigroid body is in bluish violet in Normal group mouse substantia nigra of midbrain area neuron, and densely distributed；And Parkinson's disease
Model group Substantia Nigra tigroid body significantly reduces；1 bacillus amylobacter preparation group of embodiment has clear improvement compared to model group.
4. Mice brain tissues TH contents
TH positive expressions are positioned at cytoplasm, and Parkinson's disease group mouse is decreased obviously (P compared with Normal group TH expressions
＜ 0.01), neuron fracture missing, banded structure disappears；The TH levels of 1 bacillus amylobacter preparation group of embodiment are compared with Parkinson's disease group
Rebound significantly (P ＜ 0.05), neuronal cell morphology, arrangement be improved significantly.
5. the variation of Mice brain tissues Occludin protein levels
The hippocampus positive cell number of Normal group mouse is more, and occludin expressions are high；Parkinson's disease group
Mouse positive cell number is significantly reduced compared with Normal group, and occludin expressions reduce (P ＜ 0.01)；1 butyric acid of embodiment
Bacillus preparation group mouse has clear improvement (P ＜ 0.01) compared with Parkinson's disease group.
Conclusion：
This experiment injects MPTP by mouse peritoneal and establishes parkinsonian mouse model, and mouse shows motor function barrier
Hinder and change with behaviouristics.The Parkinson disease model group mouse dead time extends, and autogenic movement ability is decreased obviously, the movement of mouse
Function and cognitive function change.After the processing of 1 bacillus amylobacter preparation of embodiment, the spontaneous activity of parkinsonian mouse is bright
Aobvious to rise, the dead time is decreased obviously, and the symptom of the Parkinson's disease of mouse is improved.Pathologyofbraintissue change is Parkinson
The seriously ill form of expression wanted, this experimental morphology it turns out that：Nissl is dyed and HE coloration results are shown, 1 butyric acid bar of embodiment
Hippocampus neuron Pathological lesions can be obviously improved after bacteria preparation processing；Propylhomoserin hydroxylase (TH) is catecholamines nerve
Rate-limiting enzyme in mediator (DA) building-up process, substantia nigra dopaminergic neuron is mainly expressed in intracerebral, is evaluation Parkinson's disease
One of standard of therapeutic effect.This experimental result is shown：TH protein expressions significantly reduce in Parkinson's disease group Mice brain tissues, warp
After the processing of 1 bacillus amylobacter preparation of embodiment, TH contents substantially increase, it is meant that 1 bacillus amylobacter preparation of embodiment is to Parkinson's disease
The reduction of the neurotransmitter DA of model mice has improvement result.
In conclusion the present invention has developed a kind of good effect, the drug for the anti-Parkinson's disease having no toxic side effect, through animal
Experimental study proves that bacillus amylobacter preparation can play the effect of anti-Parkinson's disease.
Only the preferred embodiments of the invention is not used for limiting the scope of implementation of the invention, i.e., all according to the present invention
The equivalent changes and modifications that claim is done are all that the scope of the claims of the present invention is covered.
Claims (1)
1. the Clostridium butylicum preparation of a kind of anti-Parkinson's disease, it is characterised in that the preparation method of Clostridium butylicum preparation is as follows：
(1) prepared by seed liquor：Picking single bacterium colony Clostridium butylicum (Clostridium butyricum) WZ001, deposit number are
CGMCC No.8808 are inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, and Anaerobic culturel for 24 hours, obtains seed liquor；
(2) ferment：Containing with weight percent after step (1) seed liquor has been sterilized by the inoculum concentration access of 1% weight ratio
Peptone 1-5%, yeast extract 0.5-5%, glucose 1-10%, oligofructose 0.5-2%, ammonium sulfate 0.1%, the phosphorus of meter
Acid dihydride potassium 0.05-0.3%, dipotassium hydrogen phosphate 0.05-0.3%, manganese sulfate 0.02%, magnesium sulfate 0.02% and calcium chloride
0.002%, remaining is in the fluid nutrient medium of deionized water, and anaerobic fermentations -48h for 24 hours is stood through 35 DEG C -37 DEG C；
(3) thalline is collected：Clostridium butylicum (Clostridium butyricum) WZ001 that step (2) culture obtains is fermented
Liquid, 3000rpm centrifuge 5~10min, remove supernatant, obtain bacterium mud, and bacterium suspension is made in 2~3 washings of deionized water
Liquid centrifuges 5~10min with 3000rpm again, removes supernatant fluid and obtain sediment；
(4) bacterium broken wall：By the sediment that step (3) obtains by 1: 5~1: 10 plus deionized water, the cell under 150MPa pressure
It is broken, obtain breaking-wall cell product；
(5) degreasing is eluted：The sediment that will be obtained in the step (4) carries out dissolving elution repeatedly using ethyl alcohol, 10,000rpm from
15~20min of the heart recycles sediment, and washing steps are 2~3 times, then with the washing of deionized water 3~5 times, 3000rpm centrifugations 5~
10min obtains sediment；
(6) finished product：The sediment obtained in step (5) is freeze-dried under vacuum negative pressure condition, you can obtain purity and reach
More than 95% dried powder is to get to Clostridium butylicum preparation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105055457A (en) * | 2015-08-06 | 2015-11-18 | 温州医科大学附属第二医院 | Drug for preventing and treating alzheimer's disease as well as preparation method and application of drug |
| CN105687252A (en) * | 2016-03-22 | 2016-06-22 | 温州医科大学附属第二医院 | Application of clostridium butyricum in preparation of medicines for treating and/or preventing acute liver injury |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105055457A (en) * | 2015-08-06 | 2015-11-18 | 温州医科大学附属第二医院 | Drug for preventing and treating alzheimer's disease as well as preparation method and application of drug |
| CN105687252A (en) * | 2016-03-22 | 2016-06-22 | 温州医科大学附属第二医院 | Application of clostridium butyricum in preparation of medicines for treating and/or preventing acute liver injury |
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| Title |
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| GIULIA UMBRELLO ET AL: "Microbiota and neurologic diseases: potential effects of probiotics", 《JOURNAL OF TRANSLATIONAL MEDICINE》 * |
| HUIYING WANG ET AL: "Effect of Probiotics on Central Nervous System Functions in Animals and Humans: A Systematic Review", 《J NEUROGASTROENTEROL MOTIL》 * |
| JIAMING LIU ET AL: "Neuroprotective Effects of Clostridium butyricum against Vascular Dementia in Mice via Metabolic Butyrate", 《BIOMED RESEARCH INTERNATIONAL》 * |
| JIAMING LIUA ET AL: "Sodium butyrate exerts protective effect against Parkinson"s disease in mice via stimulation of glucagon like peptide-1", 《JOURNAL OF THE NEUROLOGICAL SCIENCES》 * |
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Application publication date: 20180518 |