CN106109536B - For neurodegenerative disease or the Chinese medicine composition of nerve regneration - Google Patents
For neurodegenerative disease or the Chinese medicine composition of nerve regneration Download PDFInfo
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- CN106109536B CN106109536B CN201610629850.7A CN201610629850A CN106109536B CN 106109536 B CN106109536 B CN 106109536B CN 201610629850 A CN201610629850 A CN 201610629850A CN 106109536 B CN106109536 B CN 106109536B
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a kind of Chinese medicine compositions that can be used for treating neurodegenerative disease or nerve regneration, belong to the field of Chinese medicines.It is made of total Flavones from Rhodiola sachalinensis and urolithin.The weight ratio of the total Flavones from Rhodiola sachalinensis and urolithin is 10: 0.1-3；Preferably 10: 0.7.
Description
Technical field
The present invention relates to a kind of Chinese medicine compositions that can be used for treating neurodegenerative disease or nerve regneration, belong to Chinese medicine
Field.
Background technique
Neurodegenerative disease with protein precipitation includes a large amount of clinical symptoms.It is characterized by protein or albumen
Pathology sexual abnormality, whole body or the localized concentrations of matter segment (peptide), so as to cause precipitating or gathering, and may be by prepolymerization or low
Poly- effect becomes the structure (such as: fibrinogen) of ordered arrangement, in some instances it may even be possible to will form protein structures (such as: A Zi
The amyloid beta of the silent sick intracerebral patch in sea).
The also under cover some diseases, such as Alzheimer disease (Alzheimer ' after these pathologic symptom
Sdisease, AD) morbidity of Alzheimer disease increases with the growth at age.Currently, the hair of Alzheimer disease in the world
Sick rate male is 3.05%, and women 4.82%, and U.S. 85 years old or more dementia incidence is up to 4.72%.In western countries, Ah
Alzheimer's disease just gradually replaces stroke, occupies first of neuropathy.Patient's sum of China's Alzheimer disease has accounted for generation
First, boundary, it is contemplated that the year two thousand fifty, the elderly and man at an advanced age of China's over-65s and 80 years old or more will account for total population
35% and 22%, at that time, AD patient can reach 25,000,000.Present China 60 years old or more population is more than 10%.At present on
The drug of senile dementia is treated mainly with acetylcholinesterase inhibitor and n-methyl-D-aspartic acid receptor antagonist in city
(NMDA) based on, these drugs can improve the dementia symptom of patient to a certain extent, but cannot fundamentally prevent the state of an illness
Deteriorate, reverse disease, therefore the development for finding anti-senile dementia disease drug has caused global attention, and many has been established
Relevant bioactivity screening and appraisement system.
In addition, neurotrosis inevitably brings greatly economic and mental burden to patient, with nervus retrogression disease
Similarly damage zone can have the apoptosis phenomenon of obvious nerve cell to disease, and this phenomenon is irreversible.Therefore around
The reparation and reconstruction of neurologic defect are a great problems in Current ambient neurotrosis field.It is solved in treatment clinical course at present
The means of neurologic defect still using nerve autograft as standard procedures, but due to autologous nerve limited source and cause donor site compared with
More complication.
Rhodiola has more than 90 in the world in Crassulaceae (Crassulaceae) rhodiola (Rhodiola L) plant
Kind, China has more than 70 kinds.It is distributed in Himalayas, the Asia northwestward and North America, China's main product is in northeast, North China
The southwest and.Rhodida plant is civil to have the effect of promoting blood circulation and hemostasis, clearing lung and relieving cough mostly with all herbal medicine, for treating hemoptysis, coughing up
Diseases, the ancient times such as blood, pneumonia cough, women leukorrhea also act as strengthening by means of tonics medicine.Root of kirilow rhodiola earliest as it is medicinal see be recorded in Christian era
In Tibetan medicine's Four-Volume Medical Code in 1200, " god's medicine --- sulu Ma is preserved ", i.e., present wide arnotto red-spotted stonecrop are denoted as
(R.eurycarpa S.H.FU).Modern medical book is divided into a variety of according to rhodiola plant feature, sees as medicinal
Record the big premium scape of the Pharmacopoeia of the People's Republic of China (1985 editions) and " Sichuan Province's standard for traditional Chinese medicines " (1993 editions) records
Its (R.crenulataH.Ohba) and Ledum palustre (R.kirilowii Maxim).
Gallotannin (Ellagitannin) is in certain fruit, berry and nut (such as pomegranate, raspberry, strawberry, black
Raspberry, English walnut and almond) in the monomer polyphenol, oligomeric polyphenol and the polymerized polyphenolic that are rich in.Fruit and berry are by widely new
It is consumed when fresh, and as beverage (such as fruit juice), and it has been reported that they can promote health.
Urolithin is ellagic acid, punicalagins (PA), Tannin in Punica granatum L (punicalin) (PB), new suona horn element
(tellimagrandin) metabolin of (TL) and other gallotannins.Clinical at present and scientific research show its with it is anti-inflammatory,
Anti-oxidant and anticancer activity.
Summary of the invention
It is an object of the present invention to provide a kind of Chinese medicine groups that can be used for treating neurodegenerative disease and nerve regneration
Object is closed, is made of total Flavones from Rhodiola sachalinensis and urolithin.
In one embodiment, the weight ratio of the total Flavones from Rhodiola sachalinensis and urolithin is 10: 0.1-3；Preferably 10:
0.7。
In another embodiment, the total Flavones from Rhodiola sachalinensis refers to that root of kirilow rhodiola of the flavones ingredient higher than 90% extracts
Object, the urolithin are urolithin A.
In further embodiment of the present invention, the Chinese medicine composition is by total Flavones from Rhodiola sachalinensis, radix polygonati officinalis total saposins, the coptis
Total alkaloid and urolithin composition, total Flavones from Rhodiola sachalinensis, radix polygonati officinalis total saposins, coptis chinensis total alkaloid and urolithin weight ratio be 10
:2-5:1-4:0.1-3；Preferably 10: 3: 2: 0.7.
It is a further object to provide the Chinese medicine compositions in preparation treatment neurodegenerative disease or nerve
Application in regenerating medicine.
In one embodiment, the neurodegenerative disease is preferably Alzheimer's disease, and the nerve regneration is
Nerve regneration caused by nerve cell regeneration or promotion stem cell after referring to neural neurotrosis are gone back to the nest.The stem cell
It goes back to the nest and refers to discovery stem cell homing phenomenon, stem cell homing pair after the Various Tissues such as brain, heart and liver or organ damage
Tissue or organ damage reparation play a significant role.Stem cell homing phenomenon is equally existed after nervous system injury.
In the present invention, the total Flavones from Rhodiola sachalinensis, radix polygonati officinalis total saposins and coptis chinensis total alkaloid can directly pass through business canal
Road obtains, naturally it is also possible to be prepared by related manufacturing processes known in the art.It is specific the preparation method comprises the following steps:
Total Flavones from Rhodiola sachalinensis preparation: root of kirilow rhodiola pulverizing medicinal materials are sieved, the water refluxing extraction measured with 10 times three times, every time 1
Hour, filtering, filtrate is concentrated to give medicinal extract, by macroreticular resin LSA-10 on medicinal extract, first with n-hexane with the elution of three times column volume, so
It is eluted afterwards with 75% ethyl alcohol, collects 75% ethanol eluate, be concentrated and dried up to total Flavones from Rhodiola sachalinensis.
The preparation of radix polygonati officinalis total saposins: radix polygonati officinalis pulverizing medicinal materials are sieved, and extract filtering three times with 15 times 60 DEG C of 80% ethyl alcohol of amount,
Filtrate is concentrated to give medicinal extract, is extracted 5 times with n-butanol 1:1, merges butanol extraction liquid, upper macroreticular resin AB-8 after concentration, with
80% ethanol elution merges eluent, is concentrated and dried up to radix polygonati officinalis total saposins.
Coptis chinensis total alkaloid preparation: Rhizoma Coptidis is pulverized and sieved, after being impregnated 1 hour with 12 times of 0.1% sulfuric acid solutions of amount,
Then heating and refluxing extraction 1 hour is extracted three times repeatedly, and the macroreticular resin AB-8 of medicinal material weight 0.5% is added to filtrate for filtering
It is adsorbed, concentration filtrate to the 10% of former filtrate volume；Sodium chloride is added into concentration filtrate to 15% (weight) and to be added
Iron chloride crystallizes 48 hours, filtration precipitating, to 5% (weight) up to coptis chinensis total alkaloid after washing and drying.
Specific embodiment
Also the present invention further can be understood by embodiment, wherein the embodiment illustrates some preparations or user
Method.It is to be appreciated, however, that these embodiments do not limit the present invention.The change of the invention of currently known or further exploitation
Change is considered within the scope of the invention described herein and claimed below.
Influence of 1 Chinese medicine composition of embodiment to 42 amyloid aggregation of A β
Total Flavones from Rhodiola sachalinensis preparation: root of kirilow rhodiola pulverizing medicinal materials are sieved, the water refluxing extraction measured with 10 times three times, every time 1
Hour, filtering, filtrate is concentrated to give medicinal extract, by macroreticular resin LSA-10 on medicinal extract, first with n-hexane with the elution of three times column volume, so
It is eluted afterwards with 75% ethyl alcohol, collects 75% ethanol eluate, be concentrated and dried up to total Flavones from Rhodiola sachalinensis.
The preparation of radix polygonati officinalis total saposins: radix polygonati officinalis pulverizing medicinal materials are sieved, and extract filtering three times with 15 times 60 DEG C of 80% ethyl alcohol of amount,
Filtrate is concentrated to give medicinal extract, is extracted 5 times with n-butanol 1: 1, merges butanol extraction liquid, upper macroreticular resin AB-8 after concentration, with
80% ethanol elution merges eluent, is concentrated and dried up to radix polygonati officinalis total saposins.
Coptis chinensis total alkaloid preparation: Rhizoma Coptidis is pulverized and sieved, after being impregnated 1 hour with 12 times of 0.1% sulfuric acid solutions of amount,
Then heating and refluxing extraction 1 hour is extracted three times repeatedly, and the macroreticular resin AB-8 of medicinal material weight 0.5% is added to filtrate for filtering
It is adsorbed, concentration filtrate to the 10% of former filtrate volume；Sodium chloride is added into concentration filtrate to 15% (weight) and to be added
Iron chloride crystallizes 48 hours, filtration precipitating, to 5% (weight) up to coptis chinensis total alkaloid after washing and drying.
42 protein freeze-dried powder of 0.1mgA β, which is dissolved in 10 μ LDMSO and 543.78 μ L phosphoric acid buffer liquid systems, is configured to final concentration
For 40 μM of 42 protein stock solution of A β.12.5 μ L sample to be tested solution, 25 μ LA β, 42 protein stock solution and 12.5 μ LThT
Stock solution (concentration be 80 μM) is added in 96 hole board pores, is mixed 96 orifice plate shaker of juxtaposition and is vibrated after 15min with 90rpm in 37
DEG C carbon dioxide incubator stationary incubation 16h.After 16h, the high speed cell analysis instrument of TTP Labtech company, Britain is utilized
(Acumene X3 type) 405 channels measure its fluorescence intensity.
The calculating of the 42 albumen aggregation activity of anti-A β of active material: Vi=[(FO-Fi)/FO] × 100.Wherein, Vi is phase
To inhibiting rate, Fi is the fluorescence intensity that 42 aggregation of A β after sample to be tested is added, and F0 is the A β 42 when sample to be tested not being added
The fluorescence intensity of aggregation.
Concrete outcome is as follows:
2 Chinese medicine composition of embodiment improves senile dementia drosophila learning and memory active testing
(1) cultivation of senile dementia drosophila
Control group background drosophila of the w1118 (isoCJ1) as experiment.The drosophila for being successfully transferred to pathogenic 42 albumen of A β is
(UAS-Aβ42).The strain drosophila is hybridized by expressing Gal4 promoter drosophila with full brain, is obtained and is carried elav-
The drosophila strains of GAL4c155 (P35) and A β 42.
(2) administration of senile dementia drosophila
Three kind groups of the healthy drosophila without medicine control, disease drosophila without medicine control and the administration of disease drosophila are arranged in test.
The parent of all test drosophilas at 24 DEG C of constant temperature, raise by the fly house of constant humidity 42%RH (Relative humidity)
It supports and breeds.Pass through carbon dioxide by control group drosophila and disease group drosophila and to medicine feed group drosophila within first day after drosophila emergence
After anesthesia, the drosophila of correct character is selected in the glass tube containing food.In the administration stage, all test drosophilas are at 28 DEG C
Raising in constant temperature and the incubator of 42% constant humidity, to guarantee efficiency that drosophila takes medicine.Daily drosophila medicine feed 4 hours, from choosing fruit
Second day of fly always medicine feed by the 8th day.
Institute's medicine feed object choose prepare within fly second day and with preparation on the day of to drosophila medicine feed.Contain in addition, control group drosophila feeds
The syrup of 1%DMSO.For each Activity Index (Performance Index), 2 pipe drosophila groups are needed, are contained in every pipe
About 100 drosophilas.
1) in the training stage, about 100 or so drosophilas are packed into the training pipe for being mounted with copper mesh crossed electrode, successively
It is passed through two kinds of each 60s of smell of octanol (0CT) and methyl cyclohexanol (MCH), the fresh air of midfeather 45s.It is being passed through first
The pulse electric shock stimulation of drosophila 60V is given while kind smell (CS+) (US, pulse duration 1.5s are spaced 3.5s).It is passed through second
It does not give and shocks by electricity when kind smell (CS-).So complete a cycle of training.
2) in immediate memory (study) aptitude tests, the drosophila for completing a cycle of training is typically immediately transferred to T-Maze
Selected element, while being passed through CS+ and CS- from opposite both direction.The drosophila of two sides is received respectively after the selection of 2min
It is counted after collection, anesthesia or execution.The calculation formula of Activity Index (Performance index, PI) is as follows:
PI=[(CS-)-(CS+)]/[(CS-)+(CS+)] × 100.
OCT and MCH are used respectively as CS+ and is trained and tests, and the average value of two obtained PI is as primary real
The PI tested is used.PI=0 indicates that drosophila is selected as 50: 50 for two kinds of smells in test, i.e., without forming memory；PI=
100 indicate that the escape of drosophila whole is with the smell of electric shock, i.e. perfect memory in test.When carrying out active testing, while carrying out not
The senile dementia disease fly (P35*H29.3) of the same genetic background health fly (2U*H29.3) of medicine feed, not medicine feed feeds test medicine
The smell short-term memory defect test of senile dementia disease fly calculates separately their total learning and memory behavior index (PI).It will
Feed the same genetic background health fly (2U*H29.3) of the senile dementia disease fly learning and memory behavior index and not medicine feed of test medicine
Senile dementia disease fly (P35*H29.3) Activity Index of Activity Index, not medicine feed compares, and evaluation test drug anti-ageing year is silly
Slow-witted effect.The the senile dementia disease fly learning and memory behavior index of feeding tester relatively the high, illustrates tester anti-ageing year
Dementia effect is stronger.Compared using T inspection, the senile dementia disease fly learning and memory behavior index of feeding tester and not medicine feed
The senile dementia disease fly learning and memory behavior index of (only to the solvent of not drug containing sample), P < 0.05 are to have significant difference, P
< 0.01 is to have marked difference, and P < 0.001 is to have extremely significant difference.
Data analysis and figure displaying are handled by using GraphPad Prism 5.03
Influence of 3 Chinese medicine composition of embodiment to mouse A β model
Male mouse of kunming, skin opens an osculum at bregma after anesthesia, exposes skull, by the A β 25-35 of aging
4nmol injects telocoele, control group saline injection.Administration group gavages drug from next day, and Morris water fan is carried out after 10 days
Palace measurement, twice a day, continuous 7 days.Data acquisition and processing (DAP) is monitored automatically by image and processing system is completed.
Concrete outcome is as follows:
4 Chinese medicine composition of embodiment promotes the external BMSC of nerve trachea material to raise
Bracket and BMSC are separated using Transwell co-culture system, it is specific to be grouped are as follows:
Blank group Transwell upper chamber adds 100 μ 1BMSC cell suspensions (1 × 106A/m1), 500 μ, 1 nothing is added in lower room
Blood serum medium；
Control group Transwell upper chamber adds 100 μ 1BMSC cell suspensions (1 × 106A/ml), 500 μ 1 leaching is added in lower room
Steep the serum free medium of nerve trachea (Neurolac)；
Administration group Transwell upper chamber adds 100 μ 1BMSC cell suspensions (1 × 106A/m1), 500 μ l leaching is added in lower room
Steep the serum free medium of nerve trachea (Neurolac)；The drug of 100 μ g/L is added in the culture medium.
37 DEG C are incubated for 24 hours, and PBS is washed 3 times, and the fixed 10min of 4% paraformaldehyde wipes upper chamber with cotton swab and do not migrated film
Cell, DAPI dyes 30min, and PBS washes 3 times, and 5 visuals field, counting of taking pictures are randomly choosed under fluorescence microscope.Identical experiment weight
3 times multiple, concrete outcome is as follows:
The research of the external evoked BMSC directed differentiation nerve cell of 5 Chinese medicine composition of embodiment
Different pharmaceutical group is added in different culture dishes respectively (100 μ g/L of final concentration), it includes 1 × 10 that 1ml, which is added,6
The culture medium (10%FBS/DMEM) of a/ml BMSC, does not add any drug in control group, 37 DEG C be incubated for 8 hours after exempted from
Nestin and GFAP antibody is respectively adopted as primary antibody in the detection of epidemic disease cytochemistry, and the goat anti-mouse igg of FITC label is secondary antibody,
Calculate group of cells the positive expression rate, every group parallel test 3 times, concrete outcome is as follows:
The content of present invention merely illustrates claimed some specific embodiments, one of them or more skill
Documented technical characteristic can be combined with arbitrary one or more technical solutions in art scheme, these are combined and obtain
Technical solution also in the application protection scope, the technical solution just as obtained from these are combined is disclosed in the present invention
It is specifically recorded in content the same.
Claims (4)
1. it is a kind of for treating the Chinese medicine composition of neurodegenerative disease and nerve regneration, by total Flavones from Rhodiola sachalinensis and urinary calculi
Element composition；The weight ratio of the total Flavones from Rhodiola sachalinensis and urolithin is 10: 0.7；The total Flavones from Rhodiola sachalinensis refer to flavonoids at
Divide the gadol extract higher than 90%, the urolithin is urolithin A.
2. it is a kind of for treating the Chinese medicine composition of neurodegenerative disease and nerve regneration, by total Flavones from Rhodiola sachalinensis, radix polygonati officinalis
Total saposins, coptis chinensis total alkaloid and urolithin composition；Wherein total Flavones from Rhodiola sachalinensis, radix polygonati officinalis total saposins, coptis chinensis total alkaloid and urine
The weight ratio of Shi Su is 10: 2-5: 1-4: 0.1-3.
3. Chinese medicine composition of any of claims 1 or 2 is in preparation treatment neurodegenerative disease or nerve regneration drug
Using.
4. application according to claim 3, the neurodegenerative disease is Alzheimer's disease, and the nerve regneration is
Nerve regneration caused by nerve cell regeneration or promotion stem cell after referring to neural neurotrosis are gone back to the nest.
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