CN107233371A - The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases and medicine - Google Patents

The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases and medicine Download PDF

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CN107233371A
CN107233371A CN201710622050.7A CN201710622050A CN107233371A CN 107233371 A CN107233371 A CN 107233371A CN 201710622050 A CN201710622050 A CN 201710622050A CN 107233371 A CN107233371 A CN 107233371A
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medicine
bone
refuting
big
degenerative diseases
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彭光天
银巍
何细新
陈文礼
张翠仙
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Guangzhou University of Chinese Medicine
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Guangzhou University of Chinese Medicine
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/19Acanthaceae (Acanthus family)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/51Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

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Abstract

The present invention relates to drug field, in particular to a kind of big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases and medicine.The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases.Above-mentioned nerve degenerative diseases include at least one of Parkinson's, alzheimer disease, Huntington disease and Protein tau disease.Inventor's discovery refutes bone and its new purposes of extract greatly, and axoneuron can be prevented to occur denaturation, apoptosis;Containing the medicine for refuting bone greatly the nerve cells of HT 22 can be made to keep fit form and propagation, protect the nerve cells of HT 22, suppress the apoptosis of the nerve cells of HT 22.The medicine that the present invention is provided has preferable curative effect in the medicine for preparing treatment, auxiliary treatment or prevention of neurodegenerative diseases.

Description

It is big to refute bone answering in the medicine for preparing prevention and/or treatment nerve degenerative diseases With and medicine
Technical field
The present invention relates to drug field, prevention and/or treatment neurological are being prepared in particular to a kind of big bone of refuting Application and medicine in the medicine of property disease.
Background technology
Big bone of refuting is black leaf small-leaf bonesetting (the Justicia ventricosa of dicotyledon Acanthaceae justicia platymiscium Wall.ex Sims.) drying herb, whole year can harvest.Black leaf small-leaf bonesetting also known as bone is refuted greatly, black leaf justicia, black leaf synthetism Grass, large osteosynthesis, obliqueswollen adhatoda stem and leaf, duck son spend, force tree, refute section etc. greatly.Its is pungent;It is bitter;It is mild-natured.With promoting blood circulation and stopping pain;Stagnation resolvation connects Bone;Dispelling wind and eliminating dampness;Subdhing swelling and detoxicating.Cure mainly bruise wound swollen;Fracture;Internal lesion caused by overexertion pain in the back;Arthralgia pain due to rheumatism;Peratodynia;Lung carbuncle;Acute mastitis;Nothing Name pyogenic infections;Wound is red and swollen.Medicinal record about refuting bone greatly, first appeared in《Book on Chinese herbal medicine asks former》,《Luchuan book on Chinese herbal medicine》Also there is note Deng ancient books Carry,《The south of the Five Ridges is gathered medicinal herbs record》,《Nanning City's medicine will》, Guangzhou air force《Conventional Chinese herbal medicine handbook》Deng local medicinal herbal monograph and national Large-scale Chinese medicine monograph《Chinese book on Chinese herbal medicine》、《Dictionary of medicinal plant》、《Chinese medicine sea》Deng recording, Guangxi province Chinese medicine standard (nineteen ninety version), Guangdong Province's Chinese medicine standard (2011) and Drug Standard of Ministry of Public Health of the Peoples Republic of China Traditional Chinese medicine historical preparation 8th, the 11st, the 19th, the 20th is recorded with national standard for traditional Chinese medicines compilation orthopedics and traumatology fascicle.
The content of the invention
The medicine of prevention and/or treatment nerve degenerative diseases is being prepared it is an object of the invention to provide a kind of big bone of refuting In application and medicine, its aim to provide it is a kind of be used for prepare treatment nervous centralis degenerative disease medicament medicine;There is provided a kind of The big new purposes for refuting bone.
The present invention provides a kind of technical scheme:
The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases.
Further, the above-mentioned big extract for refuting bone is in the medicine for preparing prevention and/or treatment nerve degenerative diseases Application;
The big extract for refuting bone is mainly made by following steps:Extracted, divided after extraction using solvent by bone is refuted greatly From obtaining extract solution;Wherein, the weight ratio that bone and solvent are refuted greatly is 1:5~20.
Further, above-mentioned nerve degenerative diseases include Parkinson's, alzheimer disease, Huntington disease and tau eggs At least one of white disease.
The present invention also provides a kind of technical scheme:
A kind of medicine for preventing and/or treating nerve degenerative diseases, for preparing prevention and/or treatment nervus retrogression The medicine of disease, medicine is mainly made up of the raw material including refuting bone greatly.
Further, said medicine is mainly made by following steps:Extracted bone is refuted greatly using solvent, after extraction Isolated extract solution;Wherein, the weight ratio that bone and solvent are refuted greatly is 1:5~20.
Further, above-mentioned solvent is selected from water, methanol, ethanol, isopropanol, n-butyl alcohol, ethylene glycol, 1,2-PD, 1, One or more in ammediol, 1,3-BDO, acetone, ethyl acetate.
Further, above-mentioned solvent is that mass fraction is 30-95% ethanol waters.
Further, it is above-mentioned to refute bone ultrasonic extraction 15-60min at 30-90 DEG C greatly.
Further, also include after above-mentioned isolated extract solution to extract solution concentration, dry.
Further, it is above-mentioned that concentration is evaporated to extract solution, is concentrated under reduced pressure or film concentration;To the extract solution after concentration Carry out constant pressure and dry, be dried under reduced pressure, vacuum freeze drying or spray drying.
The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases and medicine that the present invention is provided The beneficial effect of thing is:
Inventor's discovery refutes bone and its new purposes of extract greatly, and axoneuron can be prevented to occur denaturation, apoptosis;Contain There is the big medicine for refuting bone that HT-22 nerve cells can be made to keep fit form and propagation, protect HT-22 nerve cells, suppress HT- The apoptosis of 22 nerve cells.The medicine that the present invention is provided is preparing the medicine for the treatment of, auxiliary treatment or prevention of neurodegenerative diseases There is preferable curative effect in thing.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be attached to what is used required in embodiment Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore is not construed as pair The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 shows that the medicine that the embodiment 1 of mtt assay detection is provided induces L-glutamate the protection of HT-22 cells Effect.
Fig. 2 shows that the medicine that the embodiment 1 of micro- Microscopic observation is provided induces HT-22 cells to L-glutamate Protective effect.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional production that can be obtained by commercially available purchase Product.
Bone is refuted greatly in the medicine for preparing prevention and/or treatment nerve degenerative diseases to the embodiment of the present invention below Using and medicine be specifically described.
Further, above-mentioned nerve degenerative diseases are nervous centralis degenerative disease.
Further, the above-mentioned big extract for refuting bone is in the medicine for preparing prevention and/or treatment nerve degenerative diseases Application;
The big extract for refuting bone is mainly made by following steps:Extracted, divided after extraction using solvent by bone is refuted greatly From obtaining extract solution;Wherein, the weight ratio that bone and solvent are refuted greatly is 1:5~20.Further, the weight ratio of bone and solvent is refuted greatly For 1:10-15.
Above-mentioned solvent be selected from water, methanol, ethanol, isopropanol, n-butyl alcohol, ethylene glycol, 1,2-PD, 1,3-PD, One or more in 1,3-BDO, acetone, ethyl acetate.
Further, above-mentioned nerve degenerative diseases include Parkinson's, alzheimer disease, Huntington disease and tau eggs At least one of white disease.Specifically, Protein tau disease includes frontotemporal dementia, cortico-basal degeneration, Pick's disease, progressive Supranuclear paralysis etc..
In other embodiments of the invention, above-mentioned nerve degenerative diseases also include Wilson's disease, prion disease and Other dementia include vascular dementia, ALS (ALS), Acute Stroke (neurotrosis caused by headstroke) and its His traumatic injury;Cerebrovas-cularaccident (e.g., AMD);Brain and trauma of spinal cord, peripheral nerve disease, retina Disease and glaucoma.
Nervous centralis degenerative disease is one group and denaturation, the god caused by apoptosis gradually occurs with central nervous system neurons Through degenerative disease or nervous disorders.Its cause of disease fails to understand that pathogenesis and pathological manifestations are complicated, and clinical manifestation is different mostly.Should Group disease is more common in person in middle and old age, in chronic or subacute onset, and progressive is aggravated, and the course of disease is longer, and numerous diseases and illness it Between it is overlapped, such as 10% or so Parkinson's merge dull-witted, Alzheimer, the parkinson's syndrome in Pacific Ocean Guam Deng.Due to lacking special biochemistry, iconography and pathophysiological features, the experience for often relying primarily on clinician is paid a home visit It is disconnected.In addition to a few diseases (such as Parkinson's) temporarily may improve clinical symptoms by medicine or operation, to most of disease mesh Before there is no the treatment method of special efficacy.The therapeutic strategy of such as ishemic stroke mainly includes thromboembolism treatment and Neuroprotective Therapy in Treating Acute. Thrombolytic drug Alteplase (rt-PA) be it is first be also that the thrombolytic drug for treating apoplexy is uniquely ratified by FDA so far. Although rt-PA is it is verified that acute ischemic stroke can be treated effectively, its therapeutic time window is narrow, and the mechanism of action is indefinite, There is bleeding risk, therefore only a few patients can receive rt-PA effective treatment in time.Benserazide is treatment PD Choice drug, but the severe complications such as on-off phenomenon, the deterioration of agent end occur in patient's long-term taking.Therefore neuroprotective is carried out Treatment, may postpone disease time or delay the development of the state of an illness, it has also become the focus of current research.
Neuroprotective Therapy in Treating Acute is one or more mechanism by hindering Ischemic Cascade Reaction to be related to, in starting stage protection Penumbra zone neure damage caused by ischemic, so as to prevent or postpone nerve cell death.It can realize that blood is fast by medicine Speed passes back into ischemic tissue, so as to suppress the infringement on the biochemistry level caused by ischemic, metaboilic level and cellular level.Brain lacks Blood triggers the pathological biochemistry cascade reaction of complexity, and the approach of Neuroprotective Therapy in Treating Acute is various, and link is different, the prospect of neuroprotection It is very considerable.
Found by the research and discussion of inventor, bone is refuted greatly and is preparing treatment, auxiliary treatment or prevention neurological Property disease medicine in have preferable curative effect.Refuting bone Central nervous member greatly, gradually generation denaturation, apoptosis have curative effect.Refute greatly Bone helps to protect nerve cell from cellular damage and/or resistance neurodegenerative process and the treatment of neuronal function infringement Agent or active material.
The present invention also provides a kind of technical scheme:
A kind of medicine for preventing and/or treating nerve degenerative diseases, medicine is mainly made up of the raw material including refuting bone greatly.
Further, said medicine is mainly made by following steps:Extracted bone is refuted greatly using solvent, after extraction Isolated extract solution;Wherein, the weight ratio that bone and solvent are refuted greatly is 1:5~20.
Further, above-mentioned solvent is selected from water, methanol, ethanol, isopropanol, n-butyl alcohol, ethylene glycol, 1,2-PD, 1, One or more in ammediol, 1,3-BDO, acetone, ethyl acetate.
Further, above-mentioned solvent is that mass fraction is 30-95% ethanol waters.In the present embodiment, it is quality point The ethanol water of number 95%.
Further, it is above-mentioned to refute bone ultrasonic extraction 15-60min at 30-90 DEG C greatly.
Further, also include after above-mentioned isolated extract solution to extract solution concentration, dry.
Further, it is above-mentioned that concentration is evaporated to extract solution, is concentrated under reduced pressure or film concentration;Specifically, using decompression Concentration.In preferred embodiments of the present invention, it is concentrated by evaporation using Rotary Evaporators.
Extract solution after concentration is carried out constant pressure and dry, be dried under reduced pressure, vacuum freeze drying or spray drying.To concentration Rear extract solution carry out constant pressure and dry, be dried under reduced pressure, vacuum freeze drying or spray drying.Specifically, adopt in the present invention With being dried under reduced pressure.
Refuting bone Central nervous member greatly, gradually generation denaturation, apoptosis have curative effect.The big extract for refuting bone or medicine tool There is protection neure damage, so as to prevent or postpone the effect of nerve cell death;Disease time can be postponed or delay the state of an illness Development.The big medicine for refuting bone preparation can cell keep fit form and propagation, suppress Apoptosis;The medicine is thin for nerve Born of the same parents have significant protective effect.
In addition, in other embodiments, the medicine also includes pharmaceutically acceptable carrier or auxiliary material.
For example:Diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, grape Sugar, urea, calcium carbonate, white bole, microcrystalline cellulose, alumina silicate etc.;Wetting agent and adhesive, such as water, glycerine, polyethylene glycol, Ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethylcellulose, Lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.;Disintegrant, for example dry starch, alginate, agar powder, Laminaran, sodium acid carbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, dodecyl sodium sulfate, first Base cellulose, ethyl cellulose etc.;Disintegration inhibitor, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil and fat;Absorb Accelerator, such as quaternary ammonium salt, lauryl sodium sulfate;Lubricant, such as talcum powder, silica, cornstarch, stearic acid Salt, boric acid, atoleine, polyethylene glycol etc..Tablet can also be further made to coating tablet, such as sugar coated tablet, film bag Garment piece, enteric coated tablets, or double-layer tablets and multilayer tablet.
In addition, in order to which administration unit is made into pill, various carriers well known in the art can be widely used.Such as:Grape Sugar, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talcum powder etc.;Adhesive Such as Arabic gum, bassora gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter;Disintegrant, such as agar powder, dry starch, Alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc..In order to which administration unit is made into suppository, Ke Yiguang It is general to use various carriers well known in the art.Example on carrier is, for example polyethylene glycol, lecithin, cocoa butter, senior Alcohol, the ester of higher alcohol, gelatin, semi-synthetic glyceride etc..
In addition, if desired, can also be added into pharmaceutical preparation colouring agent, preservative, spices, flavouring, sweetener or Other materials.
Further, the medicine of the embodiment of the present invention can be administered in a unit, and method of administration can be enteron aisle or non- Enteron aisle, such as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritonaeum or rectum.Form of administration for example tablet, capsule, Dripping pill, aerosol, pill, pulvis, solution, supensoid agent, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, jelly Dry powder injection etc..Can be ordinary preparation, sustained release preparation, controlled release preparation and various particulate delivery systems.In order to which unit is administered Tablet is made in formulation, can widely use various carriers well known in the art.
In addition, the dosage of the medicine of the embodiment of the present invention depends on many factors, for example, to be prevented or treat disease Sex, age, body weight and the individual reaction of the property and the order of severity of disease, patient or animal, particular compound used, administration Approach and administration number of times etc..Above-mentioned dosage with ingle dose form or can be divided into several, and such as two, three or four dosage forms give Medicine.
From the above, in administration process, the actual dose level of each active component in pharmaceutical composition can be changed, so as to The reactive compound amount of gained can effectively obtain required therapeutic response for specific patient, composition and administering mode.Dosage Level must be according to the activity of particular compound, method of administration, the order of severity for treating the patient's condition and the patient's condition of patient to be treated Selected with medical history.But, the way of this area is that the dosage of compound is wanted from less than therapeutic effect needed for obtaining The level asked starts, gradually incremental dose, until obtaining required effect.
Inventor's discovery refutes bone and its new purposes of extract greatly, and axoneuron can be prevented to occur denaturation, apoptosis;Contain There is the big medicine for refuting bone that HT-22 nerve cells can be made to keep fit form and propagation, protect HT-22 nerve cells, suppress HT- The apoptosis of 22 nerve cells.The medicine that the present invention is provided is preparing the medicine for the treatment of, auxiliary treatment or prevention of neurodegenerative diseases There is preferable curative effect in thing.
The feature and performance to the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of medicine, and the medicine is mainly prepared by following steps:
Weigh and refute bone 30g greatly, be placed in triangular flask, the ethanol waters of 300ml 95% are added into triangular flask, in 60 DEG C Ultrasonic extraction 30min, filtering, is condensed into be placed in evaporating dish after medicinal extract shape with Rotary Evaporators and is evaporated, in constant temperature oven (50 DEG C) dry to constant weight.
Embodiment 2
The present embodiment provides a kind of medicine, and the medicine is mainly prepared by following steps:
Weigh and refute bone 10g greatly, be placed in triangular flask, the ethanol waters of 50ml 30% are added into triangular flask, it is super in 30 DEG C Sound extracts 15min, and filtering is concentrated under reduced pressure into be placed in evaporating dish after medicinal extract shape and is evaporated, perseverance is dried in (50 DEG C) in constant temperature oven Weight.
Embodiment 3
The present embodiment provides a kind of medicine, and the medicine is mainly prepared by following steps:
Weigh and refute bone 10g greatly, be placed in triangular flask, the addition 200ml 60%1 into triangular flask, 2- aqueous solution of propylene glycol, In 90 DEG C of ultrasonic extraction 60min, filtering is concentrated under reduced pressure into after medicinal extract shape vacuum freeze drying to constant weight.
Embodiment 4
The present embodiment provides a kind of medicine, and the medicine is mainly prepared by following steps:
Weigh and refute bone 10g greatly, be placed in triangular flask, 100ml ethyl acetate is added into triangular flask, in 60 DEG C of ultrasonic extractions 45min, filtering is dried under reduced pressure to constant weight after film concentration.
Embodiment 5
The present embodiment provides a kind of medicine, and the medicine is mainly prepared by following steps:
Weigh and refute bone 10g greatly, be placed in triangular flask, 150ml water is added into triangular flask, in 45 DEG C of ultrasonic extraction 60min, Filtering, is condensed into be placed in evaporating dish after medicinal extract shape with Rotary Evaporators and is evaporated, and is dried to constant weight in (50 DEG C) in constant temperature oven.
Test example
The medicine that this test example is obtained to embodiment 1 carries out neuroprotection test.
The culture of 1.HT-22 nerve cells
By (three batches) HT-22 cells, with pancreatin (0.25%, 1mL), therefrom ware digests (in 1min), dispels into slender Born of the same parents' suspension, adds a small amount of hyclone FBS (100 μ L).1000rpm centrifuges 5min, removes supernatant, cell precipitation is dispersed in into 3mL In complete medium, take 1mL cell suspensions to be added in 24mL complete mediums, 10 mixings are overturned back and forth, take 0.1mL to be used for Count.It is diluted to appropriate cell concentration (8 × 104Cell/mL after), 48 orifice plates first add 50 μ L culture mediums, make culture medium thorough It is distributed in orifice plate rims.(three batches of cells, three blocks of plates, 100 μ L, 8 × 10 are planted in 48 orifice plates4cell/mL).Rotated in kind Centrifuge tube, plants a half bore and exerts oneself to blow and beat cell, be allowed to uniform, while allow cell suspension to be paved with orifice plate bottom, after all finishing Whether micro- Microscopic observation cell is scattered uniform, and cross, which shakes up, makes cell be uniformly dispersed in orifice plate bottom.Place 24 hours (cells Degrees of fusion is about 30~40%) it is used to test.
2.MTT methods are tested
It is scattered in the μ g of medicine 200 are obtained in embodiment 1 in 2mL complete mediums, changes liquid for 3 × 48 orifice plates, be MTT Experiment.Kind of 48 orifice plates of good cell are taken, are divided into Control (control group), Glutamate (glutamic acid) group, NAC (N-acetyl Cysteine) (N-acetylcystein) group, Trolox (watermiscible vitamin E) groups and the sample sets (medicine that embodiment 1 is provided Group).Every group of three multiple holes, culture medium changes liquid, per the μ L of hole 200, changes every time before liquid, blows and beats culture medium, makes dispersion of medicine.Change Complete 37 DEG C of liquid, which is incubated addition glutamic acid after 1h, makes final concentration reach 3mM (6 μ L).37 DEG C of culture 24h are placed on after shaking up.Observation is thin Born of the same parents' form, and cell survival rate (mtt assay) is detected, then with 20 μ L 37 DEG C of incubation 2h of MTT (5mg/mL) lucifuge, gently suck Liquid, adds 200 μ L DMSO, shaking table 5min to shake up per hole.Then with multi-function microplate reader calculate each light absorption value under 490nm (under Data are and measured under 490nm in the figure of face, damage group cell survival rate be about Control groups 20%).Three hole OD values are taken, are asked Average value, subtracts blank DMSO OD values, then divided by control group standardizing average values.The MTT of three batches of cells OD values Average value does statistical discrepancy with the softwares of SigmaPlot 12.5.
As shown in table 1, table 1 shows Control groups, Glutamate groups, NAC groups, Trolox groups and sample to statistical result Cell survival rate in group.
Each experimental group cell survival rate contrast of table 1 (N=3)
Experimental data in table 1 is drawn into block diagram, as a result as shown in figure 1, specifically, Fig. 1 shows mtt assay detection It is big to refute the protective effect that bone extract induces L-glutamate HT-22 cells.In figure:"+", which represents corresponding test group, phase The component answered, "-", which represents corresponding test group, does not have corresponding component.Wherein, * * P<0.01, * * * P<0.001 with glutamic acid group Compare (one-way analysis of variance, Dunnett methods).Trolox (10 μM) is used as positive control.
Shown by Fig. 1 result, the medicine that embodiment 1 is provided has significant protective effect for HT-22 nerve cells.
3. the big protective effect for refuting bone extract of micro- Microscopic observation
The μ g of medicine 20 that embodiment 1 is provided are scattered in 2mL complete mediums, and liquid is changed for 3 × 48 orifice plates.Take and plant good thin 48 orifice plates of born of the same parents, are Control groups, Glutamate groups, Glu (Glutamate)+NAC (N-acetyl cysteine) respectively Group, Glu+Trolox and Glu+ sample sets (medicine group that embodiment 1 is provided), every group of three multiple holes, culture medium changes liquid, per hole 200 μ L, are changed before liquid every time, are blown and beaten culture medium, are made dispersion of medicine.Changing addition glutamic acid after the complete 37 DEG C of incubations 1h of liquid makes end dense Degree reaches 3mM (6 μ L).37 DEG C of culture 24h are placed on after shaking up.Finally cellular morphology is observed under the microscope, and take pictures, as a result As shown in Figure 2.Fig. 2 shows that the big of micro- Microscopic observation refutes the protection that bone extract induces L-glutamate HT-22 cells Effect.As shown in Figure 2, the medicine that embodiment 1 is provided can make cell keep fit form and propagation, it is suppressed that L- Apoptosis caused by glutamate (Pidolidone).
It was found from this experimental example, the medicine that embodiment 1 is provided can cell keep fit form and propagation, suppress cell and wither Die;The medicine has significant protective effect for HT-22 nerve cells.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies Change, equivalent substitution, improvement etc., should be included in the scope of the protection.

Claims (10)

1. the big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases.
2. application according to claim 1, it is characterised in that the big extract for refuting bone is preparing prevention and/or controlled Treat the application in the medicine of the nerve degenerative diseases;
The big extract for refuting bone is mainly made by following steps:By it is described it is big refute bone and extracted using solvent, extraction Isolated extract solution afterwards;Wherein, the big bone and the weight ratio of the solvent of refuting is 1:5~20.
3. application according to claim 1 or 2, it is characterised in that the nerve degenerative diseases include Parkinson's, Ah At least one of the silent disease in Wurz sea, Huntington disease and Protein tau disease.
4. a kind of medicine for preventing and/or treating nerve degenerative diseases, it is characterised in that the medicine is main by including refuting greatly The raw material of bone is made.
5. medicine according to claim 4, it is characterised in that the medicine is mainly made by following steps:Will be described Refute bone greatly to be extracted using solvent, isolated extract solution after extraction;Wherein, the big weight ratio for refuting bone and the solvent For 1:5~20.
6. medicine according to claim 5, it is characterised in that the solvent is selected from water, methanol, ethanol, isopropanol, 1- fourths One or more in alcohol, ethylene glycol, 1,2-PD, 1,3-PD, 1,3-BDO, acetone, ethyl acetate.
7. medicine according to claim 5, it is characterised in that the solvent is that mass fraction is that 30-95% ethanol is water-soluble Liquid.
8. medicine according to claim 5, it is characterised in that refute bone ultrasonic extraction 15- at 30-90 DEG C greatly by described 60min。
9. medicine according to claim 5, it is characterised in that also include extracting to described after the isolated extract solution Liquid is concentrated, dried.
10. medicine according to claim 5, it is characterised in that concentration is evaporated to the extract solution, be concentrated under reduced pressure or Person's film is concentrated;The extract solution after concentration is carried out constant pressure and dry, be dried under reduced pressure, vacuum freeze drying or spray drying.
CN201710622050.7A 2017-07-27 2017-07-27 The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases and medicine Pending CN107233371A (en)

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