CN108261425A - A kind of functional oligose composition for mitigating Alzheimer disease and preparation method and application - Google Patents
A kind of functional oligose composition for mitigating Alzheimer disease and preparation method and application Download PDFInfo
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Abstract
The invention discloses the composition of the treatment Alzheimer disease containing functional oligose, containing ingredients such as oligofructose, bifidobacterium adolescentis active factors and hydroxybenzyl phosphate esters.The present invention relates to the applications in the drug for preparing treatment Alzheimer disease.Raw material sources of the present invention are in natural plants, and safe and non-toxic, preparation method is simple, easy to utilize.The present invention prevents Alzheimer disease from the New Century Planned Textbook for adjusting intestinal flora, has important economic society meaning and promotional value.
Description
Technical field
The present invention relates to the purposes of functional oligose, and in particular to the composition containing functional oligose is preparing treatment
Application in the drug of Alzheimer disease, belongs to field of biological pharmacy.
Background technology
Alzheimer disease (Alzheimer ' s disease, AD) is that a kind of declined with cognitive function progressive is main
The nerve retrograde affection of clinical manifestation.The disease age of onset is mostly after 65 years old, average course of disease about 8~10 years, clinical manifestation
To remember, recognizing, understanding, judging and deducing ability and social adaptation, the chronic progressive decline of personal lifestyle ability, finally
Develop into advanced dementia.Mostly hidden to attack onset, irreversibility progressive cognition dysfunction is its typical feature, and some patientss are also
It will appear personality change.The pathogenesis of AD is sufficiently complex, and the definite cause of disease of AD is unclear, but Neuropathological Study result
Prompt the disease and big intracerebral senile plaque (senile plaque, SP) and neurofibrillary tangles (neurofibrillary
Tangle, NFT) formed it is related, possible mechanism include beta-amyloyd peptide (amyloid beta, A β) deposition, inflammation damnification, oxygen
Change stress, radical damage etc..
AD is a kind of neurodegenerative disease gradually developed, and the cause of disease is not also fully aware of, it may be possible to h and E because
Plain collective effect induce as a result, and old-age group is its important risk factor.AD in the elderly of studies have shown that over-65s at present
Incidence be about 12%, incidence about 50%, the AD of the elderly AD of 85 years old or more has become current gerontology institute face
One of the problem of sternness the most faced.With the continuous renewal of microecology of intestinal tract theory and constantly carrying for microecology technology
Height adjusts the balance of intestinal microecology, it has also become one of important channel of improvement and treatment neuropsychiatric disease.It is good for as human body
The intestinal flora of health " barometer " and the various diseases of the mankind, such as enteric microorganism and colon cancer, oral microorganism and cancer of pancreas
Etc. correlations be recognized, and the variation of its intestinal flora is often ignored in the treatment of AD, for AD rear intestinal flora ecologic structures
Whether change still unknown.But in clinical AD patient often with the disease of the intestinal disorders such as halitosis, constipation, diarrhea
Shape, according to current microecology theory, the intestinal microecology that can deduce these patients has occurred and that change.In recent years, with
The progress of brain-gut axis theory, the structure and function of intestinal flora is for maintaining normal cognitive ability to have important meaning
Justice.Also, clinical and Research of Animal Model for Study is found, the structure and function existing defects of intestinal flora in AD enteron aisles.Therefore, from
The New Century Planned Textbook of adjusting intestinal flora finds new AD therapeutic schemes or means are likely to become the new strategy of AD treatments.
Due to its pathogenesis complexity, there is presently no highly effective therapies.At present, the medicine of AD is main
Including two major class：Anticholinesterase (such as Aricept) and excitatory amino acid NMDA (N-methyl-D-aspartate) receptor
Antagonist (such as easy times of Shen).Only have 5 drugs to go through to list so far.These drugs are all symptomatic treatment, and therapeutic effect is not
Ideal, and it is big to take toxic side effect for a long time, while its is expensive, is easily recurred after drug withdrawal, can not reverse or effect a radical cure AD.
In addition, although the medicine that a large amount of clinical test is dedicated to targeted inhibition A β level is researched and developed, these drugs it is long-term
Validity and safety are not still verified effectively.
Oligosaccharide is also known as oligosaccharides, is the carbohydrate of low molecular weight, usually passes through glucosides key connection by 2~10 monosaccharide
It forms, ordinary oligosaccharide and functional oligose can be divided into.Functional oligose is digestible oligosaccharide, main to include newborn ketone
Sugar, oligofructose and galactooligosaccharide, are referred to as " prebiotics ".Prebiotics refer to can be by one kind of enterobacteriaceae selective use
Component changes flora composition with activity so as to play beneficial effect to host health.Oligofructose by 1~4 fructosyl with β-
2,1 the key connections ketose (GF2) of formation, Nystose (GF3), sugarcane sugar (GF4) on the D-Fructose base of sucrose
It is the widest prebiotics of research with the mixture of sugarcane fruit six sugared (GF5).Oligosaccharide cannot be digested enzyme point in stomach and small intestine
Solution, is utilized by beneficial bacterium selective fermentation in colon, can increase the content of caecum short chain fatty acids, can also increase colon pancreas height
The secretion of blood glucose element sample peptide 1 and 5-HT.It yet there are no the correlative study that oligofructose is used to treat to Alzheimer disease
And report.
Invention content
The defects of it is an object of the invention to overcome the prior art and deficiency, oligofructose and bifidobacterium adolescentis activity because
Son is applied on the disease of Alzheimer disease improves, and then develops a kind of safe, effective treatment Alzheimer disease
Composition.
A kind of composition for being used to treat Alzheimer disease, the composition includes oligofructose, bifidobacterium adolescentis is lived
Sex factor and hydroxybenzyl phosphate ester, each component weight ratio are：
Oligofructose：60-90 parts by weight；
Bifidobacterium adolescentis active factors：10-30 parts by weight；
Hydroxybenzyl phosphate ester：5-10 parts by weight；
The preparation method of the bifidobacterium adolescentis active factors is as follows：
(1) prepared by seed liquor：Picking single bacterium colony bifidobacterium adolescentis (Bifidobacterium adolescentis)
LJM-001, deposit number are CGMCC No.4090, are inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel
For 24 hours to get to bifidobacterium adolescentis LJM-001 seed liquors；
(2) it ferments：Containing with weight hundred after step (1) seed liquor has been sterilized by the inoculum concentration access of 1% weight ratio
Point than meter peptone 1-5%, yeast extract 0.5-5%, glucose 1-10%, L-cysteine hydrochloride 0.01-1%,
Ammonium sulfate 0.1%, potassium dihydrogen phosphate 0.05-0.3%, dipotassium hydrogen phosphate 0.05-0.3%, manganese sulfate 0.01-0.05%, sulfuric acid
Magnesium 0.001-0.005% and calcium chloride 0.001-0.005%, remaining is in the fluid nutrient medium of deionized water, through 35 DEG C -37 DEG C
Stand anaerobic fermentation for 24 hours~48h；
(3) thalline is collected：The bifidobacterium adolescentis LJM-001 zymotic fluids that step (2) culture is obtained, 3,000rpm centrifugations
5~10min removes supernatant, obtains bacterium mud, and bacterial suspension is made in 2~3 washings of deionized water, again with 3,
000rpm centrifuges 5~10min, removes supernatant fluid and obtains 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) finished product is lyophilized：The sediment obtained in step (4) is freeze-dried under vacuum negative pressure condition, you can obtain
Purity reaches more than 95% dried powder to get to bifidobacterium adolescentis active factors.
Bifidobacterium adolescentis (Bifidobacterium adolescentis) LJM-001 of the present invention, in 2010 08
The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 17th, is referred to as CGMCC (addresses：North
The institute 3 of Jing Shi Chaoyang Districts North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is
Bifidobacterium adolescentis (Bifidobacterium adolescentis), deposit number are CGMCC No.4090.
Since present invention firstly discloses made using oligofructose, bifidobacterium adolescentis active factors as medicament active composition
The application of standby treatment Alzheimer disease drugs, therefore, medicament is made with auxiliary material combination, as long as the medicament is used to treat A Er
Ci Haimo diseases, all belong to the scope of protection of the present invention.
Advantages of the present invention and effect：
The invention firstly discloses oligofructose, bifidobacterium adolescentis active factors to treat Alzheimer for key component
The purposes of disease, and the preparation method of the cell activity ingredient of bifidobacterium adolescentis active factors is provided.The present invention is relative to oligomeric
Fructose exclusive use is more excellent in the effect of anti-Alzheimer disease.
Specific embodiment
Embodiment 1
The present invention includes each component of following weight for treating Alzheimer disease composition：
| Composition | Weight (mg) |
| Oligofructose | 88 |
| Bifidobacterium adolescentis active factors | 6 |
| Hydroxybenzyl phosphate ester | 6 |
The preparation method of bifidobacterium adolescentis active factors
(1) prepared by seed liquor：Picking single bacterium colony bifidobacterium adolescentis (Bifidobacterium adolescentis)
LJM-001, deposit number are CGMCC No.4090, are inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel
For 24 hours to get to bifidobacterium adolescentis LJM-001 seed liquors；
(2) it ferments：Containing with weight hundred after step (1) seed liquor has been sterilized by the inoculum concentration access of 1% weight ratio
Point than meter peptone 3%, yeast extract 2%, glucose 8%, L-cysteine hydrochloride 0.05%, ammonium sulfate 0.1%,
Potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, manganese sulfate 0.02%, magnesium sulfate 0.002% and calcium chloride 0.002%,
In the remaining fluid nutrient medium for deionized water, anaerobic fermentation is stood for 24 hours through 35 DEG C；
(3) thalline is collected：The bifidobacterium adolescentis LJM-001 zymotic fluids that step (2) culture is obtained, 3,000rpm centrifugations
5min removes supernatant, obtains bacterium mud, and 3 washings of deionized water are made bacterial suspension, are centrifuged again with 3,000rpm
10min removes supernatant fluid and obtains sediment；
(4) bacterium broken wall：By the sediment that step (3) obtains by 1: 5 plus deionized water, cell is broken under 150MPa pressure
It is broken, obtain breaking-wall cell product；
(5) finished product is lyophilized：The sediment obtained in step (4) is freeze-dried under vacuum negative pressure condition, you can obtain
Purity reaches more than 95% dried powder to get to bifidobacterium adolescentis active factors.
Described bifidobacterium adolescentis (Bifidobacterium adolescentis) LJM-001, in 08 month 2010
China Committee for Culture Collection of Microorganisms's common micro-organisms center was deposited in 17th, is referred to as CGMCC (addresses：Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is blueness
Spring Bifidobacterium (Bifidobacterium adolescentis), deposit number are CGMCC No.4090.Oligofructose is purchased from
Guangdong Jiangmen quantum high-tech.
Embodiment 2
The present invention includes each component of following weight for treating Alzheimer disease composition：
| Composition | Weight (mg) |
| Oligofructose | 80 |
| Bifidobacterium adolescentis active factors | 10 |
| Hydroxybenzyl phosphate ester | 10 |
The wherein preparation method of bifidobacterium adolescentis active factors
(1) prepared by seed liquor：Picking single bacterium colony bifidobacterium adolescentis (Bifidobacterium adolescentis)
LJM-001, deposit number are CGMCC No.4090, are inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel
For 24 hours to get to bifidobacterium adolescentis LJM-001 seed liquors；
(2) it ferments：Containing with weight hundred after step (1) seed liquor has been sterilized by the inoculum concentration access of 1% weight ratio
Point than meter peptone 5%, yeast extract 1%, glucose 7%, L-cysteine hydrochloride 0.5%, ammonium sulfate 0.1%,
Potassium dihydrogen phosphate 0.2%, dipotassium hydrogen phosphate 0.05%, manganese sulfate 0.05%, magnesium sulfate 0.003% and calcium chloride 0.003%,
In the remaining fluid nutrient medium for deionized water, anaerobic fermentation is stood for 24 hours through 37 DEG C；
(3) thalline is collected：The bifidobacterium adolescentis LJM-001 zymotic fluids that step (2) culture is obtained, 3000rpm centrifugations
6min removes supernatant, obtains bacterium mud, and 3 washings of deionized water are made bacterial suspension, are centrifuged again with 3000rpm
8min removes supernatant fluid and obtains sediment；
(4) bacterium broken wall：By the sediment that step (3) obtains by 1: 5 plus deionized water, cell is broken under 150MPa pressure
It is broken, obtain breaking-wall cell product；
(5) finished product is lyophilized：The sediment obtained in step (4) is freeze-dried under vacuum negative pressure condition, you can obtain
Purity reaches more than 95% dried powder to get to bifidobacterium adolescentis active factors.
Described bifidobacterium adolescentis (Bifidobacterium adolescentis) LJM-001, in 08 month 2010
China Committee for Culture Collection of Microorganisms's common micro-organisms center was deposited in 17th, is referred to as CGMCC (addresses：Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is blueness
Spring Bifidobacterium (Bifidobacterium adolescentis), deposit number are CGMCC No.4090.Oligofructose is purchased from
Guangdong Jiangmen quantum high-tech.
3 zoopery of embodiment
APP/PS1 transgenosis AD mouse models are randomly divided into model group, 1 processing group of embodiment, oligofructose processing group,
And using wild type C57BL/6J mouse as Normal group.
First, materials and methods
1. behaviouristics detects
1.1 new object identification experiments：Memory and exploring ability are investigated, experiment point carries out for 5 days, mouse is enabled to know within the 1st, 2 day
Other housing environment is put into two identical cubes on the the 3rd, 4 day and enables its familiar objects in the case, is within the 5th day two the previous day
One in a cube changes the volume black centrum similar with height into, and it is (i.e. old to explore cube respectively by comparing mouse
Object) and the time length of centrum (i.e. new object) analyze the memory capability of mouse.
1.2 spontaneous activities are tested：Spontaneous activity box is an enclosed environment, without any light, uses infrared analysis.It measures
The indexs such as move distance, the movement velocity of rat in spontaneous activity box in 30min.
2.Western blot：Dislocation of cervical vertebra method put to death mouse, take out brain tissue rapidly, move to -80 DEG C preserve in case
Western blot are tested.Transferring film, primary antibody are incubated, and lower 4 DEG C of shaking table jog overnight, washes film, and secondary antibody is incubated, and washes film, chromogenic reaction
And exposure, gray scale scanning is carried out to egative film, gray analysis is carried out using Image J softwares.
3. pathology and immunohistochemistry：Brain tissue paraformaldehyde is perfused, fixed, dehydration, transparent, paraffin embedding, and slice is exempted from
The operating procedure by specification of epidemic disease histochemical staining carries out, the operation of congo red staining conventional method.
4. statistical analysis：Normal distribution data information is compared using t inspections, and non-normal data data uses non-ginseng
Number Wilcoxon is examined, and P ＜ 0.05 represent that difference is statistically significant.
Two, results：
1. behaviouristics detects：In spontaneous activity experiment, compared with Normal group, the movement total distance of AD model groups is shown
It writes and reduces (P ＜ 0.05), the activity time significantly shortens (P ＜ 0.05), after oligofructose processing, moves total distance and activity time
Improved (P ＜ 0.05), 1 processing group of embodiment improves more obvious.
In new object identification experiment, compared with Normal group, AD model group cognitive functions significantly decline (P ＜
0.05), after oligofructose is handled, cognition dysfunction is obviously improved (P ＜ 0.01), and 1 processing group of embodiment improves cognition work(
Energy obstacle is more excellent.
2. the congo red staining of hippocampal tissue：Normal group group neurons of hippocampus CA 1 is clear in structure complete, nucleus
Structure is normal；Mouse hippocampal neuron neuron beta-amyloid protein deposition showed increased can be observed in AD model groups；It is individually low
Hippocampus of mice area neuron beta-amyloid protein deposition significantly reduces after Fructooligosaccharides processing, and 1 processing group of embodiment improves better than low
Fructooligosaccharides processing group.
3.Western blot：Compared with Normal group group, AD model group BDNF and PSD-95 protein expression reduce (P
＜ 0.05 or P ＜ 0.01), compared with AD model groups, independent oligofructose processing group BDNF and PSD-95 protein expression increases (P
＜ 0.05 or P ＜ 0.01), 1 processing group BDNF and PSD-95 protein expression of embodiment increases, better than the processing of independent oligofructose
Group.
Conclusion：
Compared with prior art, the prevention Alzheimer disease that the present invention has developed a kind of good effect, has no toxic side effect
Drug, proved through animal experiment study, the present invention treated mouse Nerve cognitive function be improved significantly, brain tissue
PSD-95 and bdnf protein expression significantly raising, improve AD Pathologic changes.
In conclusion only the preferred embodiments of the invention, is not used for limiting the range of implementation of the invention, i.e., it is all
The equivalent changes and modifications done according to scope of the present invention patent are all that the scope of the claims of the present invention is covered.
Claims (1)
1. a kind of for treating the composition of Alzheimer disease, the composition includes oligofructose, bifidobacterium adolescentis activity
The factor and hydroxybenzyl phosphate ester, each component weight ratio are：
Oligofructose：60-90 parts by weight；
Bifidobacterium adolescentis active factors：10-30 parts by weight；
Hydroxybenzyl phosphate ester：5-10 parts by weight；
The preparation method of the bifidobacterium adolescentis active factors is as follows：
(1) prepared by seed liquor：Picking single bacterium colony bifidobacterium adolescentis (Bifidobacterium adolescentis) LJM-
001, deposit number be CGMCC No.4090, be inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel for 24 hours, i.e.,
Obtain bifidobacterium adolescentis LJM-001 seed liquors；
(2) it ferments：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%, L-cysteine hydrochloride 0.01-1%, the sulfuric acid of meter
Ammonium 0.1%, potassium dihydrogen phosphate 0.05-0.3%, dipotassium hydrogen phosphate 0.05-0.3%, manganese sulfate 0.01-0.05%, magnesium sulfate
0.001-0.005% and calcium chloride 0.001-0.005%, remaining for deionized water fluid nutrient medium in, it is quiet through 35 DEG C -37 DEG C
Put anaerobic fermentation for 24 hours~48h；
(3) thalline is collected：The bifidobacterium adolescentis LJM-001 zymotic fluids that step (2) culture is obtained, 3000rpm centrifugations 5~
10min removes supernatant, obtains bacterium mud, and bacterial suspension is made in 2~3 washings of deionized water, again with 3000rpm from
5~10min of the heart removes supernatant fluid and obtains 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) finished product is lyophilized：The sediment obtained in step (4) is freeze-dried under vacuum negative pressure condition, you can obtain purity
Reach more than 95% dried powder to get to bifidobacterium adolescentis active factors.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113891716A (en) * | 2019-08-06 | 2022-01-04 | 上海绿谷制药有限公司 | Methods of treating alzheimer's disease by modulating amino acid levels |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113891716A (en) * | 2019-08-06 | 2022-01-04 | 上海绿谷制药有限公司 | Methods of treating alzheimer's disease by modulating amino acid levels |
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