CN100335066C - Muscle refining extract, its preparation method and application - Google Patents
Muscle refining extract, its preparation method and application Download PDFInfo
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- CN100335066C CN100335066C CNB2005100339666A CN200510033966A CN100335066C CN 100335066 C CN100335066 C CN 100335066C CN B2005100339666 A CNB2005100339666 A CN B2005100339666A CN 200510033966 A CN200510033966 A CN 200510033966A CN 100335066 C CN100335066 C CN 100335066C
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Abstract
The present invention relates to a muscle refining extract, a preparation method thereof and application thereof. The preparation method of the muscle refining extract comprises the following steps: pulverized muscles are dissolved in a physiological saline or phosphate buffer solution and centrifugated to obtain supernatant fluid; a filter membrane is degermed and centrifugally separated to obtain supernatant fluid; lower layer liquid is obtained by an ultrafiltration membrane; glucose gel chromatography is carried out, and the obtained chromatography eluent passes through the filter membrane and is degermed; the mixture passes through the ultrafiltration membrane to obtain the muscle refining extract. The obtained muscle refining extract has the actions for enhancing the neural stem cell activity, promoting the neural stem cell proliferation and replenishing reduced nerve fibers, and can be used for preparing medicine for treating and postponing nerve retrograde diseases, such as alzheimer's disease, etc.
Description
Technical field
The present invention relates to field of medicaments, relate to class muscle refining extract and preparation method thereof specifically;
The invention still further relates to described muscle refining extract and be used for the treatment of application in the medicine of neurodegenerative diseases in preparation.
Background technology
Alzheimer (Alzheimer ' s disease, AD) be senile dementia, be a kind of be the cerebral retrogressive disease of feature with carrying out property cognitive dysfunction and memory impairment.Show as the hypophrenia of comprehensive and lasting, comprise going down of memory, computing power, abstract thinking ability and language function, emotion and dystropy occur, disablement and independent living ability.The early stage behavioristics of this disease changes into slight disturbance of memory or personality change, runs down in 5～10 years, serious dementia symptom finally occurs, can't take care of oneself.This latent quality---the ability of memory, reasoning, abstract and language of having deprived the tool human characteristic of victim with destructive brain degenerative disease of attacking.Senile plaque (senile plaque, SP), neurofibrillary tangles (neurofibrillary tangle, NFT) and cerebral neuron death appears, synapse is lost and reactive glial cells hyperplasia etc. is the main pathological change of AD.
The senile dementia curative of using is mostly based on cholinesterase inhibitor, as tacrine (Tacrine), Huperzine A-Zhulin Antun (Huperzine A), aricept (Aricept), Exelon (Exelon), donepezil (Donepezil) etc. at present.Yet these medicine long-term effects are unsatisfactory, and reason is that senile dementia not merely is the problem of a cholinergic neurotransmitter system, but polytype neuronal damage and death.Increasing result of study shows, senile dementia is because inherited genetic factors and environmental factors, through complicated pathological process, finally cause neuronal damage, cause that neuron is connected with neuronic---the damage even the disappearance of synapse, the collapse of neutral net, thus the memory cognitive dysfunction caused.The neuron volume enlarges and the synapse disappearance can be summed up as I type malnutrition aixs cylinder.The conjugate spirals silk that phosphorylation tau (microtubule-associated protein tau) forms in the born of the same parents, promptly NFT can be summed up as II type malnutrition aixs cylinder.Senile plaque also exists in normal old people's brain, and just quantity shows that than senile dementia patient much less senile plaque is the final pathological product that various factors causes pathophysiological change.The senile dementia cause of disease is unclear at present, though some cause of disease hypothesis have been proposed, all can not be convincing fully, its therapeutic strategy does not obtain the confirmation of clinical experiment yet.The cause and onset of disease mechanism of AD is very complicated, is to be the disease or the clinical syndrome of the multi-pathogenesis of feature with gradual Cognitive function damage.Even if single etiological treatment is useful, because the existence of its multi-pathogenesis, curative effect can be not very good yet.In addition when gerontal patient discovery has memory disorder, generally all entered in the disease, late period, the cause of disease plays a role, this moment, pathological change expanded to cerebral cortex from Hippocampus and entorhinal cortex, brain has slight atrophy, and most of neurons are in damaged condition or dead, and pathology basically forms.This moment etiological treatment probably late (curative effect is not obvious).Except AD, other neurodegenerative diseases, the problem as PD, pick disease, Huntington disease and malnutrition lateral sclerosis exist equally.Therefore, early stage etiological treatment is comparatively simple, and curative effect is better, and the etiological treatment in late period is comparatively complicated, and except that general etiological treatment, as the neuron of untimely additional death, curative effect is very poor.So how to protect existing function of neurons and promote in the brain self stem cell division, breed and be converted into normal neurons, the strategy that becomes the crucial of treatment senile dementia and get a good chance of with the neuron of replenish lost.
At present, in birds, Rodents, primates and the human brain of growing up, all find the existence of neural stem cell.This shows in high vertebrates adult, can produce newborn neuron, and traditional neuron idea that can only reduce of can not regenerating has been proposed challenge.After these stem cell were implanted in the body of mice or rat, they can form new blood vessel, can make softer sclerotin hardening, even can active migration remove to seek the spinal column of damage and brain and they are repaired.The cell transplantation experiment shows that the stem cell in the brain can form the myocyte, and medullary cell then can form neurocyte.As if these results of study strengthened such conclusion: from the stem cell of different tissues, when being stimulated, can change original growth track by appropriate signals, and away from them original development models and form, a kind of new cell of bud into.If similar phenomenon also can take place in human brain, this will mean if use some sources of skin or bone marrow to be easy to stem cell, can be used for treating neurological degenerative disease.This also is why in recent years the research of stem cell worldwide be subjected to the reason greatly paid attention to.Neural stem cell has potential clinical value because of it, has become one of focus of current life science research.Neural stem cell is a kind of pluripotency undifferentiated stem cell that self renewal, self replication and differentiation become neuron, astrocyte and oligodendrocyte that has.Have now found that neural stem cell does not exist only in the nervous system of growth, also be present in the Adult Mammals central nervous system of (comprising the people), as the dentate gyrus of Hippocampus, the chamber inferior segment of tricorn.The discovery of neural stem cell and externally successfully cultivate, for the treatment belt of central nervous system disease (as nerve injury, neurodegenerative diseases) comes life and dawn, also the research for stem cells hyperplasia, differentiation mechanism provides new material and method.If can impel these stem cell divisions and the neuron that is divided into various functions, or transplant the part stem cell to patient's brain, will improve the neuronic regeneration capacity of patient greatly, recover its original function.Medicine to division and the differentiation of stem cell are worked is treatment brain injury and neurodegenerative diseases such as AD, and parkinson (Parkinson ' s disease, PD) provide new approach.Therefore, searching promotes that the effective ingredient of cell proliferation of nerve cord and differentiation is significant from natural drug or body itself.
Summary of the invention
The object of the present invention is to provide a kind of muscle refining extract, this muscle refining extract can promote cell proliferation of nerve cord and differentiation.
Another object of the present invention provides the preparation method of described muscle refining extract.
Another object of the present invention is to provide described muscle refining extract to be used for the treatment of application in the neurodegenerative diseases medicine in preparation.
The preparation method of muscle refining extract of the present invention comprises the steps:
(1) muscle of normal saline or phosphate buffer dissolving through pulverizing, the centrifuging and taking supernatant;
(2) after the filter membrane degerming, supernatant is got in centrifugalize;
(3) get subnatant by ultrafilter membrane;
(4) glucose gel chromatography, the chromatography eluent that obtains is through the filter membrane degerming;
(5) cross ultrafilter membrane, obtain muscle refining extract.
The present invention does not have strict restriction to the consumption of normal saline or phosphate buffer, dissolves muscle fully and is as the criterion to reach.The muscle of the best that the investigative test of inventor's process is found and the weight in grams of normal saline or phosphate buffer: volume milliliter ratio is 1: 5～10, adopts the ultrafilter membrane of 3-300KD, and described filter membrane aperture is 0.22 μ m.
The rotating speed of the described centrifugalize of step (2) is 3000-7000 rev/min, and temperature is 1-4 ℃.
The muscle refining extract of described method preparation can be used for preparing the application in the medicine that is used for the treatment of neurodegenerative diseases.
Described neurodegenerative diseases is an Alzheimer.
Described neurodegenerative diseases is parkinson disease.
The inventor finds that through investigative test muscle refining extract has the effect for the treatment of neurodegenerative diseases such as Alzheimer significantly.Adopt In vitro culture mice neural stem cell, set up the cell model of cell proliferation of nerve cord, add muscle refining extract, the result shows that the various dose group all has proliferation function to neural stem cell, MTT shows that administration group cytoactive obviously increases, neural ball count shows the administration group more than the blank group, and statistics has significant difference, and is the most obvious with high dose group.Experiment is expelled to muscle refining extract in the kunming mice tricorn in the body, and the result shows neural stem cell had significant proliferation in the administration group brain, and the blank group does not almost have stem cell to increase.More than experiment shows that muscle refining extract has the neural stem cell of raising activity, promotes the effect of its propagation, thereby can reach treatment and the purpose that delays neurodegenerative diseases such as AD.
Beneficial effect of the present invention: the senile dementia curative of using is mostly based on cholinesterase inhibitor at present, and the therapeutic effect of these medicines is undesirable.The present invention has the neural stem cell of raising activity through a large amount of muscle refining extracts that experimental results show that, promote its propagation, replenish the neuronic effect that reduces, thereby can reach treatment and the purpose that delays neurodegenerative diseases such as Alzheimer, through the test avirulence.Its therapeutic effect is remarkable, has a extensive future.
The specific embodiment
The preparation of embodiment 1 muscle refining extract
The male Mus cervical vertebra dislocation of will growing up is put to death, peel off and get hindlimb muscle, at 4 ℃ with 0.9% normal saline rinsing 3 times.Separating muscle is shredded, in 1: 5 ratio (quality: volume ratio) add 0.9% normal saline.Use refiner homogenate.Centrifugal (5000rpm, 40min, 4 ℃) remove precipitation, get supernatant.Further low-temperature and high-speed centrifugal (13500g, 40min, 4 ℃) is got supernatant.Cross 300KD ultrafilter membrane (13500g, 40min, 4 ℃) and get subnatant.Cross 0.22 μ m filter membrane degerming ,-20 ℃ frozen.The swelling of sephadex chromatography gel: add 200ml 0.9% normal saline in the 10g Sephadex G75 dry powder, boiling water bath 3h, cooling is removed fine grained, and is removed gas.The dress post: (1) fills it up with earlier eluant in post, checks whether leak, and gets rid of bubble in the post, closes liquid outlet, makes the volume of eluent in the post account for 15% of cumulative volume; (2) swollen Sephadex G75 is slowly injected in the post, avoid bubble to produce; (3) install and stop 10min, open liquid outlet, discharge excessive eluent; (4) keep 2～3cm eluent on the glue, mix up flow velocity (3ml/min), balance.The preparation of sample and last sample: it is tangent that eluent is dropped to the bath gel surface, sample volume is 5% of a bed volume, carefully be added in above the glue along tube wall, open column outlet, infiltrate the gel bed fully to sample, wash once along tube wall with the 2ml eluent, weighing apparatus is compressed and washed take off bottle and link to each other with post upper end and carry out continuous eluting.Eluting; Collect eluent.Cross 0.22 μ m filter membrane degerming.High speed centrifugation is got supernatant, crosses the 3KD ultrafilter membrane and obtains muscle refining extract, through the test avirulence.
The preparation of embodiment 2 muscle refining extracts
The male Mus cervical vertebra dislocation of will growing up is put to death, peel off and get hindlimb muscle, at 4 ℃ with 0.9% normal saline rinsing 3 times.Separating muscle is shredded, in 1: 10 ratio (quality: volume ratio) add 0.9% normal saline.Use refiner homogenate.Centrifugal (5000rpm, 40min, 4 ℃) remove precipitation, get supernatant.Further low-temperature and high-speed centrifugal (13500g, 40min, 4 ℃) is got supernatant.Cross 100KD ultrafilter membrane (13500g, 40min, 4 ℃) and get subnatant.Cross 0.22 μ m filter membrane degerming ,-20 ℃ frozen.The swelling of sephadex chromatography gel: add 200ml 0.9% normal saline in the 10g Sephadex G75 dry powder, boiling water bath 3h, cooling is removed fine grained, and is removed gas.The dress post: (1) fills it up with earlier eluant in post, checks whether leak, and gets rid of bubble in the post, closes liquid outlet, makes the volume of eluent in the post account for 15% of cumulative volume; (2) swollen Sephadex G75 is slowly injected in the post, avoid bubble to produce; (3) install and stop 10min, open liquid outlet, discharge excessive eluent; (4) keep 2～3cm eluent on the glue, mix up flow velocity (3ml/min), balance.The preparation of sample and last sample: it is tangent that eluent is dropped to the bath gel surface, sample volume is 5% of a bed volume, carefully be added in above the glue along tube wall, open column outlet, infiltrate the gel bed fully to sample, wash once along tube wall with the 2ml eluent, weighing apparatus is compressed and washed take off bottle and link to each other with post upper end and carry out continuous eluting.Eluting; Collect eluent.Cross 0.22 μ m filter membrane degerming.High speed centrifugation is got supernatant, crosses the 3KD ultrafilter membrane and obtains muscle refining extract, through the test avirulence.
The preparation of embodiment 3 muscle refining extracts
The male Mus cervical vertebra dislocation of will growing up is put to death, peel off and get hindlimb muscle, at 4 ℃ with 0.9% normal saline rinsing 3 times.Separating muscle is shredded, in 1: 7 ratio (quality: volume ratio) add 0.9% normal saline.Use refiner homogenate.Centrifugal (5000rpm, 40min, 4 ℃) remove precipitation, get supernatant.Further low-temperature and high-speed centrifugal (13500g, 40min, 4 ℃) is got supernatant.Cross 3KD ultrafilter membrane (13500g, 40min, 4 ℃) and get subnatant.Cross 0.22 μ m filter membrane degerming ,-20 ℃ frozen.The swelling of sephadex chromatography gel: add 200ml 0.9% normal saline in the 10g Sephadex G75 dry powder, boiling water bath 3h, cooling is removed fine grained, and is removed gas.The dress post: (1) fills it up with earlier eluant in post, checks whether leak, and gets rid of bubble in the post, closes liquid outlet, makes the volume of eluent in the post account for 15% of cumulative volume; (2) swollen Sephadex G75 is slowly injected in the post, avoid bubble to produce; (3) install and stop 10min, open liquid outlet, discharge excessive eluent; (4) keep 2～3cm eluent on the glue, mix up flow velocity (3ml/min), balance.The preparation of sample and last sample: it is tangent that eluent is dropped to the bath gel surface, sample volume is 5% of a bed volume, carefully be added in above the glue along tube wall, open column outlet, infiltrate the gel bed fully to sample, wash once along tube wall with the 2ml eluent, weighing apparatus is compressed and washed take off bottle and link to each other with post upper end and carry out continuous eluting.Eluting; Collect eluent.Cross 0.22 μ m filter membrane degerming.High speed centrifugation is got supernatant, crosses the 3KD ultrafilter membrane and obtains muscle refining extract, through the test avirulence.
The muscle refining extract that embodiment 4 embodiment 1 obtain is to the influence of the neural stem cell growth conditions of In vitro culture
Neural stem cell is inoculated in 24 well culture plates, be divided at random in dosage group (5%) in blank group, positive controls, extract low dose group (10%), the extract, extract high dose group (2.5%) the adding culture hole, behind 4d, use mtt assay to detect its activity.Result such as table 1:
Table 1
| Group | Quantity | The OD value |
| Dosage group (.5%) high dose group (5%) in the solvent matched group positive controls low dose group (2.5%) | 6 6 6 6 6 | 0.114±0.017 0.181±0.042 ** 0.171±0.018 ** 0.201±0.036 ** 0.250±0.021 ** |
*Compare P＜0.01. with the solvent matched group
Show that muscle refining extract has Nutrition to neural stem cell, improve its survival rate.Can be used for treatment and delay neurodegenerative diseases such as Alzheimer.
The muscle refining extract that embodiment 5 embodiment 1 obtain is to the proliferation of neural stem cells effect of In vitro culture
Neural stem cell is inoculated in 24 well culture plates, be divided at random in dosage group (5%) in blank group, positive controls, extract low dose group (2.5%), the extract, extract high dose group (10%) the adding culture hole, behind 7d, adopt neural ball count method.Result such as table 2:
Table 2
| Group | Quantity | The NP number |
| Dosage group (.5%) high dose group (10%) in the solvent matched group positive controls low dose group (2.5%) | 6 6 6 6 6 | 2±1 46±6 ** 26±4 ** 33±2 ** 52±3 ** |
*Compare P＜0.01. with the solvent matched group
Show that muscle refining extract has short proliferation function to neural stem cell.Can be used for treatment and delay neurodegenerative diseases such as Alzheimer.
Embodiment 6
Take by weighing the muscle refining extract lyophilized powder 10g that embodiment 1 obtains, add 120g starch and 50g dextrin and 2% sodium carboxymethyl cellulose solution 40ml, abundant mixing is made soft material in agitator.Soft material is made 20 order granules with oscillating granulator; 60 ℃ of airpillow-dry 1h of fluid bed; dried granule adds magnesium stearate 3g after with high speed pelletizing machine granulate; hydroxypropyl cellulose 3g; in V-Mixer, fully mix; make 500 of capsules with automatic capsule filling machine, aluminum-plastic packaged, promptly get the capsule of muscle refining extract lyophilized powder.Can be used for treatment and delay neurodegenerative diseases such as Alzheimer.
Embodiment 7
Take by weighing the muscle refining extract lyophilized powder 3g that embodiment 1 obtains respectively, add starch 200g, place quick granulator, add 2% sodium carboxymethyl cellulose solution 25ml, marumerization is made 12 order granules.Granule is 60 ℃ of dry 4h in baking oven, with oscillating granulator 12 eye mesh screen granulate, are sub-packed in the aluminium foil bag by the 5g/ bag, and sealing promptly gets the granule of muscle refining extract.Can be used for treatment and delay neurodegenerative diseases such as Alzheimer.
Embodiment 8
Take by weighing the muscle refining extract lyophilized powder that embodiment 1 obtains, the tween 80 that adds water for injection 1000ml and 0.2% makes dissolving, add 0.1% active carbon, boil 30min, put cold, the filtering active carbon, fill is to the 2ml ampoule, sealing by fusing, hot pressing steam is sterilized, and promptly gets the injection of muscle refining extract lyophilized powder.Can be used for treatment and delay neurodegenerative diseases such as Alzheimer.
The muscle refining extract that embodiment 9 embodiment 1 obtain is to the effect of mice tricorn inferior segment proliferation of neural stem cells
1 method:
Mice is divided into 4 groups at random, solvent matched group (artificial cerebrospinal fluid), positive controls (rhFGF), muscle refining extract low dose group (2.5mgmL
-1) and muscle refining extract high dose group (25mgmL
-1).Pentobarbital sodium anesthetized mice (40mgkg
-1), be fixed on the brain solid positioner, carry out intracerebroventricular injection.Coordinate: 3mm before the chimney of back, 1.5mm is opened on the side, inserting needle 1.5mm.The bilateral tricorn is respectively injected 5 μ l.One-sided inject time 5min, let the acupuncture needle remain at a certain point 5min.Fixing behind the operation 7d with the perfusion of 4% paraformaldehyde, paraffin embedding, continuous coronal section (5 μ m are thick) is got one for per 4, and each specimen is got 10 sections, does Nestin and Musashi immunohistochemical staining respectively according to conventional method, and every kind of antibody dyes 5 sections.Haematoxylin is redyed, and dehydration is transparent, the neutral gum mounting.
2 results
2.1 morphological observation: high dose group: the visible positive cell comparatively dense of tricorn inferior segment (SVZ), Cytoplasm dyeing is dark-brown, and negative cells is owing to haematoxylin dyeing is blue.Low dose group: chamber inferior segment positive cell number is less.Two groups positive cell form and positive controls are similar.The solvent matched group: do not have obvious positive cell substantially, cell is blueness.Cell all is dark-brown on the tricorn locular wall, an anti-false positive results that causes of not washed off for slicing edge in the SABC process.
2.2 morphometric and stereologic analysis: under light microscopic (* 250), every section is got 3 visuals field of tricorn inferior segment at random and is carried out cell counting and calculate positive cell occurrence rate (positive cell/total cellular score) under this visual field.Use SPSS mathematical statistics software and each group data carried out t check in variance analysis and the group, result such as table 3:
Cell positive rate (%) behind table 3 immunohistochemical staining
| The group rate | Animal (only) | Nestin antibody staining positive rate | Musashi antibody staining sun |
| Solvent matched group positive controls low dose group high dose group | 10 10 10 10 | 6.42±3.75 46.94±8.75 ** 21.93±5.34 **△ 44.38±8.14 ** | 5.21±2.42 43.94±9.81 ** 22.40±4.48 **△ 40.39±7.75 ** |
*Compare P＜0.01 with the solvent matched group;
△Compare P＜0.05 with positive controls
Compare with the solvent matched group, all the other 3 groups all have significant difference.Compare high dose group P＞0.05 with positive controls; Low dose group is P＜0.05 then, illustrates that two group differences have statistical significance, and the low dose group effect is not as good as positive controls and high dose group.Nestin and Musashi coloration result basically identical.Can be used for treatment and delay neurodegenerative diseases such as Alzheimer.
Claims (4)
1, a kind of preparation method of muscle refining extract is characterized in that comprising the steps:
(1) the muscle with normal saline or phosphate buffer dissolving through pulverizing, the centrifuging and taking supernatant;
(2) after the filter membrane degerming, supernatant is got in centrifugalize;
(3) get subnatant by ultrafilter membrane;
(4) glucose gel chromatography, the chromatography eluent that obtains is through the filter membrane degerming;
(5) cross ultrafilter membrane, obtain muscle refining extract.
2, preparation method according to claim 1, it is characterized in that the weight in grams of muscle and normal saline or phosphate buffer: volume milliliter ratio is 1: 5～10, adopts the ultrafilter membrane of 3-300KD, described filter membrane aperture is 0.22 μ m.
3, preparation method according to claim 1 and 2 is characterized in that the rotating speed of the described centrifugalize of step (2) is 3000-7000 rev/min, and temperature is 1-4 ℃.
4, the muscle refining extract of claim 1, the preparation of 2 or 3 described methods is used for the treatment of application in the medicine of neurodegenerative diseases in preparation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1216768A (en) * | 1998-10-29 | 1999-05-19 | 吉林大学 | Biologic antibiotic peptide, and method for preparing same |
| CN1399553A (en) * | 1999-07-26 | 2003-02-26 | 坎-菲特生物药物有限公司 | Composition containing muscle-derived active agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1216768A (en) * | 1998-10-29 | 1999-05-19 | 吉林大学 | Biologic antibiotic peptide, and method for preparing same |
| CN1399553A (en) * | 1999-07-26 | 2003-02-26 | 坎-菲特生物药物有限公司 | Composition containing muscle-derived active agents |
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