CN105147740A - Pharmaceutical composition for improving cerebral ischemic injury as well as preparation method and application of pharmaceutical composition - Google Patents
Pharmaceutical composition for improving cerebral ischemic injury as well as preparation method and application of pharmaceutical composition Download PDFInfo
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- CN105147740A CN105147740A CN201510490151.4A CN201510490151A CN105147740A CN 105147740 A CN105147740 A CN 105147740A CN 201510490151 A CN201510490151 A CN 201510490151A CN 105147740 A CN105147740 A CN 105147740A
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- clostridium butyricum
- cerebral ischemia
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Abstract
The invention belongs to the field of life science, relates to application of a probiotic preparation in preparing a medicine for improving cerebral ischemic injury, and in particular relates to novel pharmaceutical application of clostridium butyricum in improving cerebral ischemic injury. The invention discloses a probiotic preparation which is capable of cerebral ischemic injury, wherein the probiotic preparation includes medicines, health products and drinks which are free from any toxic or side effect. The invention creatively adopts intestinal flora as a therapeutic target and creates a new model of improving cerebral ischemic injury; and the pharmaceutical composition not only can reduce disease symptoms and relieve nerve cell injury, but also can bring about good social and economic benefits; therefore, the pharmaceutical composition is worthy of popularization and application.
Description
Technical field
The present invention relates to the novelty teabag of Clostridium butyricum, being specifically related to Clostridium butyricum preparing the application improved in cerebral ischemia compositions, belonging to field of biological pharmacy.
Background technology
Apoplexy is also known as apoplexy, can by apoplexy, sudden cardiac arrest, the many factors such as the thromboembolism occurred during Cardio-pulmonary Operation or hypotension cause, cerebral ischemia can cause brain cell injury, and after recovering cerebral blood flow Reperfu-sion, its ischemia injury increases the weight of on the contrary further, the cerebral tissue change caused by cerebral blood flow supply obstacle, after ischemia, secondary injury is the main cause of ischemic cerebrovascular aggravation, the brain tissue impairment that cerebral ischemia causes is a very important pathophysiological mechanism in its pathogenic process, its high incidence, high disability rate and high case fatality rate, the sickness rate of apoplexy has different according to the difference in area, about 136 ~ 4,86/,100,000.Current apoplexy has become the deputy cause of death of Chinese, brings huge misery and heavy financial burden to human lives.
The pathophysiological mechanism of cerebral ischemia reperfusion injury is complicated, it is a cascade reaction fast that cerebral ischemia re-pouring produces damage to brain cell, comprise a lot of link, the damage of such as reactive oxygen free radical, intracellular calcium overload, leukocyte and the infringement of cytokine, the neurotoxic effect of excitatory amino acid and neuronal metabolic obstacle etc.At present, the mechanism of ischemic cerebrovascular mainly comprises apoptosis, inflammatory reaction, oxidative stress, mitochondrial function exception etc.Cerebral ischemia is often with the generation of a large amount of oxygen-derived free radicals, inflammatory factor, and neurocyte edema, the apoptosis of neurocyte and necrosis, make patient's worsening prognoses, even cause death, alleviates the key link that ischemical reperfusion injury is Treatment of Ischemic Stroke.Cerebral ischemia causes mitochondrial oxidative stress and dysbolismus and neuronal apoptosis in close relations, brain cell mitochondrial dysfunction and ROS generate that to increase may be that one of the main inducing with development occurs central nervous system (CentralNervousSystem, CNS) ischemic damage and reperfusion oxidative damage.A large amount of oxygen-derived free radicals produces, and damage dna, finally causes the apoptosis of neuronal cell.Cerebral ischemia can cause the Ca of cerebral tissue
2+and Na
+at intracellular accumulation, and promote the release of irritability glutamic acid, oxygen-derived free radicals, platelet and endotheliocyte can be activated, then cause microvascular blocking.Cerebral ischemia is impaired also can cause inflammatory reaction, and the inflammatory reaction that cerebral ischemia is brought out can cause the microglia in the neutrophilic granulocyte in blood, Monocytes/Macrophages and cerebral tissue, astrocyte and endotheliocyte to discharge inflammatory cytokine at damage field.The immunocyte of brain cell and infiltration has secreted a large amount of cytokines and regulatory factor, IL-1, the inflammatory factors such as IL-6, TNF-α, and the destruction of leukocytic infiltration and BBB can be promoted, inflammatory factor can raise expression of adhesion molecule, produce oxygen active substance (reactiveoxygenspecies, ROS), destroy mitochondrial electron transport chain, and affect genetic transcription, thus induction secondary injury.Cerebral ischemia Brain Injury After is a complicated pathophysiological process, finally causes neuronal death, neuronal apoptosis and cerebral ischemia close relation.Apoptosis is present in cerebral ischemia reperfusion injury, is free radical the further developing of lipid peroxidation injury cell when cerebral hypoxia ischemia.The sickness rate of ischemic cerebrovascular, prevalence, mortality rate, disability rate are all very high, in recent years, that studies Imaging in Patients with Cerebral Ischemia Disease along with medical circle deepens continuously, and has had been found that a lot of new Therapeutic Method and medicine, but the curative effect for the treatment of brain ischemia medicament clinically unsatisfactory.
" probiotic bacteria " is active microorganism, effectively can improve alteration of intestinal flora, produce good health effect, be prepared into multiple probiotics preparation at present, comprise medicine, health product or beverage etc.Intestinal microbial population is as the important ingredient of body, its effect is not only confined to gastrointestinal tract, intestinal microbial population-intestinal-brain the axle also consisted of 3 approach (immunity, vagus nerve, neuroendocrine approach) of brain intestinal axle produces tremendous influence to brain function, makes us have behavior and emotion and completely newly and is more comprehensively familiar with.A lot of nervous system disease, as infantile autism, depression, all also have with enteric flora disturbance and close contact with brain-gut axis relevant, brain-gut axis is one and is present in central nervous system and unifies the bi-direction interactive system of the complexity between intestinal microbial population.Improve cerebral ischemia by probiotic bacteria regulating intestinal canal flora and have no relevant report both at home and abroad.Clostridium butyricum (Clostridiumbutyricum) is present in the intestinal of soil, animal and human's body, it is Gram-positive anaerobic bacillus(cillus anaerobicus), gastric acid can be tolerated and enter intestinal, the regeneration of secretion intestinal mucosa and the important nutrient butyric acid (butanoic acid) repaired, the intestinal mucosa of repairing damage, diminish inflammation, nutrition intestinal, to the intestinal tract disease that a variety of causes comprises enterobacterial infection, chemotherapy of tumors, surgical operation etc. caused, there is good curative effect, up to the present, there is not yet the research application report that Clostridium butyricum improves cerebral ischemia.
Summary of the invention
The object of the invention is the defect and the deficiency that overcome prior art, the object of this invention is to provide a strain Clostridium butyricum and improving the application in cerebral ischemia.Clostridium butyricum of the present invention is prepared into probiotics preparation, can cerebral ischemia be improved.
The invention provides the Clostridium butyricum that a strain has treatment of vascular dementia.
Clostridium butyricum of the present invention is: Clostridium butyricum (Clostridiumbutyricum) WZ001CGMCCNo.8808.
Clostridium butyricum of the present invention (Clostridiumbutyricum) WZ001, in on 01 22nd, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is referred to as CGMCC (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is Clostridium butyricum (Clostridiumbutyricum) WZ001, and deposit number is CGMCCNo.8808.
Clostridium butyricum of the present invention (Clostridiumbutyricum) WZ001 has following microbial characteristic:
(1) colonial morphology: be creamy white, circle is slightly convex, and edge is irregular, diameter 1 ~ 3mm, and surface is gloss slightly, has bubble to produce in fluid medium.
(2) individual morphology: be G
+bacillus cereus, thalline is in straight or knee shape, and 0.6 ~ 1.2 × 3.0 ~ 7.0 μm, end is round, single, paired, short chain, accidental long filament shape thalline.
(3) physiological and biochemical property: maltose (+); Mannitol (+); Raffinose (+); Lactose (+); Ribose (+); Starch (+).
Well-grown under anaerobism, does not grow in aerobic environment.Optimum growth temperature 35 ~ 40 DEG C; Minimum growth temperature 25 ~ 28 DEG C; The highest 43 ~ 45 DEG C; Growth optimum pH 6.5 ~ 7.0; PH4.5 ~ 5.0 or 8.0 ~ 8.5 do not grow.
Clostridium butyricum WZ001 of the present invention makes pharmaceutical composition as main active ingredient, improves cerebral ischemia.
Clostridium butyricum WZ001 of the present invention refers to microorganism alive.
The present invention is using the Clostridium butyricum of effective dose as medicament active composition, according to certain preparation process, add the conventional excipient substance such as excipient, flavoring agent, antiseptic, lubricant, adhesive, make the dosage form that any one is suitable for using clinically, as dosage forms such as tablet, capsule, granule, powder, oral liquid, enemas.
Clostridium butyricum compositions of the present invention can be that Clostridium butyricum makes viable bacteria probiotics preparation as active ingredient separately, also can be that Clostridium butyricum and other non-probiotic active compositions combine and make active bacteria formulation, to play synergistic therapeutic action, improve vascular dementia prevention effect.
Indication effective dose of the present invention refers to can not lower than 1 × 10 according to total viable count that the solid live bacteria preparation made as active ingredient alone or in combination described in above comprises using Clostridium butyricum
7cFU/g, generally 1 × 10
8more than CFU/g, can reach 1 × 10
12cFU/g or 1 × 10
12more than CFU/g.
The preferred preparation process of the present invention is inoculate strain in first liquid medium within, carries out cultivating or multistage amplification cultivation, then that liquid culture is centrifugal, collects bacterium mud, and described bacterium mud is dry, sieve, obtain dry mycopowder.Dry for gained mycopowder is mixed with pharmaceutical carrier, makes final dosage form.Preparation process is not limited to of the present invention, and other known preparation process is all passable.
Clostridium butyricum of the present invention, when making any one dosage form, all has the effect improving cerebral ischemia.
Because the present invention makes public for the first time with Clostridium butyricum, to be medicament active composition preparing the application improved in cerebral ischemia damnification medicament; therefore; being that active ingredient and auxiliary material combination make medicament with Clostridium butyricum, as long as this medicament is for improving cerebral ischemia, all belonging to protection scope of the present invention.
Up to the present, the present inventor selects Clostridium butyricum to improve cerebral ischemia first, evident in efficacy, and with the increase of dosage, curative effect is strengthened, and does not find any toxic and side effects, is disclose the novelty teabag that Clostridium butyricum improves cerebral ischemia first.Have no the research report of Clostridium butyricum treatment vascular dementia, the object of this invention is to provide a kind of new pharmaceutical applications of Clostridium butyricum, namely Clostridium butyricum is preparing the application improved in cerebral ischemia compositions, and said composition comprises medicine, health product and beverage etc.
Advantage of the present invention and effect:
Clostridium butyricum is play therapeutical effect from many aspects to vascular dementia, mainly comprises the following aspects:
1, Clostridium butyricum supplements beneficial bacteria of intestinal tract, and suppresses harmful intestinal tract bacteria, promotes intestinal beneficial bacteria growing, recovers intestinal microbial population balance.
2, Clostridium butyricum is target spot by intestinal, by brain intestinal axle, in brain, plays neuroprotective, improves cerebral ischemia effect.
3, the present invention can improve brain microcirculation, anti-apoptosis amount.
Detailed description of the invention
The preparation of Clostridium butyricum preparation first prepares mycopowder, then dose corresponding adjuvant as required and make corresponding dosage form, the present invention is specifically prepared as example with oral clostridium butyricum active bacteria powder, the preparation method of probiotic bacteria is described, the preparation method of other dosage form is omitted, illustrate no longer one by one, concrete preparation dosage form is not limited to method described below.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The Isolation and ldentification of embodiment 1 Clostridium butyricum (Clostridiumbutyricum) WZ001 bacterial strain
Using the feces of a healthy young people as sample separation, on modified MRS agar culture medium, under 37 DEG C of anaerobic conditions, 48h is coated with separation and Culture, obtain Clostridium butyricum of the present invention (Clostridiumbutyricum) WZ001, be accredited as Clostridium butyricum to belong to, in on 01 22nd, 2014 in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is Clostridium butyricum (Clostridiumbutyricum), preserving number is CGMCCNo.8808.
The formula of modified MRS agar culture medium: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K2HPO42g, MgSO47H2O0.5g, MnSO44H2O0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween 80 1ml, agar 15g, distilled water 1L, adjust pH to 7.0,115 DEG C of sterilizings 15 minutes.
Clostridium butyricum of the present invention (Clostridiumbutyricum) WZ001 has following microbial characteristic:
(1) colonial morphology: be creamy white, circle is slightly convex, and edge is irregular, diameter 1 ~ 3mm, and surface is gloss slightly, has bubble to produce in fluid medium.
(2) individual morphology: be G
+bacillus cereus, thalline is in straight or knee shape, and 0.6 ~ 1.2 × 3.0 ~ 7.0 μm, end is round, single, paired, short chain, accidental long filament shape thalline.
(3) physiological and biochemical property: maltose (+); Mannitol (+); Raffinose (+); Lactose (+); Ribose (+); Starch (+).
Well-grown under anaerobism, does not grow in aerobic environment.Optimum growth temperature 35 ~ 40 DEG C; Minimum growth temperature 25 ~ 28 DEG C; The highest 43 ~ 45 DEG C; Growth optimum pH 6.5 ~ 7.0; PH4.5 ~ 5.0 or 8.0 ~ 8.5 do not grow.
Clostridium butyricum of the present invention (Clostridiumbutyricum) WZ001, in on 01 22nd, 2014 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is referred to as CGMCC (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is that Clostridium butyricum belongs to, and deposit number is CGMCCNo.8808.
The preparation of embodiment 2 Clostridium butyricum (Clostridiumbutyricum) WZ001 probiotics preparation
1, seed liquor preparation: the mono-bacterium colony of picking Clostridium butyricum (Clostridiumbutyricum) WZ001, be inoculated in 50mL modified MRS agar culture medium, pH is 7.0 ± 0.5,37 DEG C, and Anaerobic culturel 24h, is seed liquor;
The formula of modified MRS agar culture medium: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K2HPO42g, MgSO47H2O0.5g, MnSO44H2O0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween 80 1mL, agar 15g, distilled water 1L, adjust pH to 7.0,115 DEG C of sterilizing 15min.
2, fermentation culture: the inoculum concentration with 5% is seeded in new modified MRS agar culture medium, 37 DEG C, Anaerobic culturel 24h, terminates to cultivate;
3, collect thalline: with 12,000rpm, 10min condition, remove fermented supernatant fluid, collect bacterium mud for subsequent use;
4, mycopowder preparation: collect bacterium mud, weigh, add freeze drying protectant (defatted milk powder and sodium glutamate), lyophilizing, porphyrize by 1: 1 (w/v), cross 100 mesh sieves, be prepared into viable bacteria mycopowder ,-4 DEG C save backup.
5, finished product preparation
According to the viable count of Clostridium butyricum mycopowder, add various adjuvant with needs in proportion, make final dosage form, make viable count be not less than 1 × 10
7cFU/g or 1 × 10
7cFU/mL.
Embodiment 3 improves the zoopery of cerebral ischemia
Object: observe probiotics preparation of the present invention on improve Antioxidant Indexes and brain morphology in cerebral ischemia, brain change and probiotics preparation of the present invention application on the impact improving cerebral ischemia mice.
1. materials and methods:
Medicine: Clostridium butyricum (Clostridiumbutyricum) WZ001 mycopowder is 1 × 10 containing viable count
8cFU/mL, preparation method is specifically shown in embodiment 2.
Animal: ICR mice 30, body weight 18-22g.
2. test method:
2.1 cerebral ischemia mouse model preparations: adopt the one-sided common carotid artery obturation (rUCCAO) in the permanent right side, after mouse anesthesia, be fixed on operating-table, be separated and sew up and carry out right carotid with permanent little silk thread, the body temperature of mice remains on 37 DEG C of constant temperature.After skin closure, mice gets back in cage and feeds recovery normally.Model success mice 20, is divided into model group (10) and Clostridium butyricum treatment group (10).Only open skin not connect bundle and be classified as Sham-operated control group (10).
2.2 Clostridium butyricum (Clostridiumbutyricum) WZ001 treats: cerebral ischemia mouse model success latter 24 hours after, oral administration gavage Clostridium butyricum WZMC1016, concentration 1 × 10
9cFU/ days/mice, once a day, continuous 4 time-of-weeks.
3. result of the test
3.2 couples of cerebral ischemia Mice brain tissues SOD, the impact of MDA
Result is known, and cerebral ischemia model group Mice brain tissues SOD vigor comparatively sham operated rats obviously reduces (P < 0.01); MDA content comparatively sham operated rats obviously increases (P < 0.05); Treatment group Mice brain tissues SOD vigor of the present invention all comparatively cerebral ischemia model group mice significantly improves (P < 0.01), and MDA content all comparatively model group obviously reduces (P < 0.01) above result and shows that medicine of the present invention has obvious antioxidation.
3.3 impacts on cerebral morphology
HE coloration result shows, and rats in sham-operated group brain tissue cell is neat and orderly, and organizational structure is complete, and neuron karyon is clear, and kernel is obvious, and endochylema uniform coloring is bright or light dye, and after birth is complete, without obvious edema and cell death.The visible obviously neuron of cerebral ischemia group occurs that swelling, pyknosis and dissolving are even dead, central ischemic zone part vanished cell, structural deterioration, decreased number, arrangement disorder, cell volume is not of uniform size, and form is irregular, part cell pyknosis triangularity, part cell cavity sample becomes, karyon engrain, nuclear membrane is unclear, and kernel is not obvious.After Clostridium butyricum WZ001 treats 4 weeks, neurocyte form is normal, arranges regular, the red dye of part endochylema homogenizing, and nuclear membrane is clear, and without corresponding change, cellularity is normal, obviously alleviates the pathological change of cerebral ischemia.
3.4 impacts on cerebral circulation blood volume
Result shows, drug medication hindbrain blood flow of the present invention obviously raises, difference tool significance (P < 0.01) compared with cerebral ischemia model group, shows medicine of the present invention and has certain effect increasing cerebral circulation blood flow.
Result of the test shows, and cerebral ischemia re-pouring causes cerebral tissue structure function generation major injury and apoptosis, and prompting Clostridium butyricum WZ001 has protective effect to cerebral ischemia reperfusion injury.
Claims (4)
1. Clostridium butyricum is preparing the application improved in cerebral ischemia compositions, and described compositions comprises medicine, health product and beverage.
2. Clostridium butyricum is preparing the application improved in cerebral ischemia compositions according to claim 1, it is characterized in that Clostridium butyricum (Clostridiumbutyricum) WZ001, this bacterial strain was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCCNo.8808 on 01 22nd, 2014.
3. Clostridium butyricum is preparing the application improved in cerebral ischemia compositions according to claim 1, it is characterized in that Clostridium butyricum refers to that the microorganism of survival is individual.
4. Clostridium butyricum, preparing the application improved in cerebral ischemia compositions, is characterized in that Clostridium butyricum compositions is not less than 1 × 10 with effective dose viable count according to claim 1
7the compositions that CFU/g Clostridium butyricum is made as active ingredient.
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| CN201510490151.4A CN105147740A (en) | 2015-08-06 | 2015-08-06 | Pharmaceutical composition for improving cerebral ischemic injury as well as preparation method and application of pharmaceutical composition |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357142A (en) * | 2007-08-05 | 2009-02-04 | 青岛东海药业有限公司 | Use of Clostridium butyricum in preparing medicine composition for preventing and treating cerebrovascular disease |
| WO2012073170A2 (en) * | 2010-11-29 | 2012-06-07 | Vetalis Sarl | Galenic composition suitable for administration to a non-human animal, uses thereof, and associated methods |
-
2015
- 2015-08-06 CN CN201510490151.4A patent/CN105147740A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357142A (en) * | 2007-08-05 | 2009-02-04 | 青岛东海药业有限公司 | Use of Clostridium butyricum in preparing medicine composition for preventing and treating cerebrovascular disease |
| WO2012073170A2 (en) * | 2010-11-29 | 2012-06-07 | Vetalis Sarl | Galenic composition suitable for administration to a non-human animal, uses thereof, and associated methods |
Non-Patent Citations (2)
| Title |
|---|
| L. ZHANG ET AL.,: ""Effects of Clostridium butyricum on growth performance, immune function,"", 《POULTRY SCIENCE》 * |
| 闫述翠等: ""酪酸梭菌在小白鼠肠道内的动态变化研究"", 《中国新药杂志》 * |
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