CN110403932A - A kind of pharmaceutical composition and its application containing withaferin A - Google Patents
A kind of pharmaceutical composition and its application containing withaferin A Download PDFInfo
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Abstract
The present invention provides a kind of pharmaceutical composition and its application containing withaferin A, belong to medicine and biology techniques field, including withaferin A and Quercetin, and the mass ratio of the withaferin A and Quercetin is 1:(1~1500).In the present invention; neurodegenerative disease can be prevented and treated under the mass ratio of withaferin A and Quercetin; neuron is protected; improve the distribution of substantia nigra tyrosine hydroxylase positive neuron and form; treat neuroinflamation; it is not only substantially reduced respective dosage, can more be achieved the effect that better than exclusive use.
Description
Technical field
The invention belongs to medicine and biology techniques field more particularly to a kind of pharmaceutical composition containing withaferin A and its
Using.
Background technique
A kind of disease that neurodegenerative disease is body neuronal structure or function is gradually lost and causes, including pa gold
Gloomy disease, Alzheimer's disease, Huntington's disease etc.；The current this kind of disease cause of disease is still not clear, and there is no effective healing means,
And seriously threaten the quality of life of patient.
Alzheimer disease (Alzheimer ' s disease, AD) is a kind of the age-related chronic of onset concealment
Carry out the central nervous system degenerative disease of sexual development.Clinically with memory disorders, aphasia, appraxia, agnosia, visual space technical ability damage
Evil, execution dysfunction and the performance of the generalized dementias such as personality and behavior change are characterized, and the cause of disease is unknown so far.With the whole world
Aging of population, A Erci are also less than disease incidence and significant ascendant trend are presented, meanwhile, because the body and mind that it seriously endangers patient is strong
Health and life quality bring heavy burden to family and society, become serious social public health problem, are current
Urgent problem.
Parkinson's disease (Parkinson's disease, PD) also known as shaking plasy, are a kind of minds for being common in person in middle and old age
Through system degenerative diseases.Clinically using static tremor, bradykinesia, myotonia and postural balance obstacle as main feature.Its
Disease incidence is higher, is not only incremented by the age, is also in become younger, and have the high-incidence trend of globalization.The main pathology of Parkinson's disease changes
It is dead to become the denaturation of substantia nigra of midbrain (substantia nigra, SN) dopamine (dopamine, DA) serotonergic neuron, black substance-line
The denaturation of shape body (striatum) dopaminergic pathway, striatal dopamine Transmitters significantly reduce, and dopamine mediator reduces
Degree is positively correlated with patient symptom severity；Pathologic marker Lewy body in the dopaminergic neuron cytoplasm of remaining
The formation of (lewy's body), main component are alpha-synapse nucleoprotein (α-synuclein).The Parkinson's disease cause of disease is still endless
All clear Chu, it is now recognized that withering with inherent cause, environmental factor, oxidative stress, excitatory toxicity, mitochondria dysfunction and cell
It is related to die equal many factors.
Parkinson's disease clinical diagnosis at this stage relies primarily on medical history, clinical symptoms and sign, and detection is aided in animal model
Black substance, the variation of corpus straitum tyrosine hydroxylase enzyme level.Wherein, tyrosine hydroxylase (tyrosine hydroxylase, TH) is
The rate-limiting enzyme of catecholamines active material biosynthesis, plays a significant role in the adjusting of dopamine biosynthesis, therefore,
In vivo, the variation of activity and expression quantity, directly affects the biology of L-3,4 dihydroxyphenylalanine amine to the enzyme especially in black substance, corpus straitum
Synthesis.Therefore, tyrosine hydroxylase enzyme level is evaluation black substance, in corpus straitum, the common finger of dopaminergic neuron level variation
Mark.In MPTP-induced Parkinson disease mice model, substantia nigra dopaminergic neuron apoptosis is mainly induced with MPTP, simulates clinical Parkinson's disease hair
Disease symptoms；And Lewy body main component alpha-synapse nucleoprotein is overexpressed in mouse black substance and is realized.
At this stage, the purpose of neurodegenerative disease primary treatment is the progress for delaying disease, the symptom for controlling disease；Treatment
Method is by drug reduction of patient Development process and extent；Most of patients symptom can be eased because of drug therapy,
But the progress of lesion cannot be prevented, and often is forced to be discontinued because side effect is excessive, or because long term usage drug effect weakens.Therefore, one is found
Kind effectively prevents, treats the drug of neurodegenerative disease, promotes life in patients, is current urgent problem.
Withaferin A (withaferinA) is the natural steroid extracted from India's Ayurveda traditional herbal medicines withania somnifera
Class compound, is soluble in organic solvent, is insoluble in water.Research has shown that withaferin A has anti-inflammatory, antitumor, neuroprotection, the heart
Vascular protection effect etc., and the symptoms such as anxiety, cognitive disorder, insomnia can be effectively relieved.2016 one be published in " Nature
Medicine " on article point out that withaferin A can significantly resist the obesity of high fat diet induction, and improve the Portugal of obesity mice
Grape sugar stable state.Show that withaferin A is effectively reduced reactive oxygen species, adjusts mitochondrial function, apoptosis involvement in addition, also having been reported that
Regulation etc., these factors and Parkinson's disease morbidity are closely related, and prompting withaferin A may be effectively to prevent, treat Parkinson's disease
Drug.
Quercetin (quercetin) chemistry entitled 3,3', 4', 5,7- pentahydroxyflavone, be present in many plants flower,
In leaf, fruit, pharmacological action is extensive, has the effects that anticancer, anti-inflammatory, cardiovascular system protection.But because of its slightly water-soluble and
The characteristics such as high dose toxicity is stronger, therefore clinically seldom it is applied to the prevention and treatment of the nervous system disease.
Field of medicaments personnel can inevitably inhibit cell activity when using withaferin A and Quercetin, the problem without
Method solves, in addition, because it with certain toxicity and has slight Central nervous depressant, so, use liquor-saturated eggplant element as
Drug therapy neurodegenerative disease, playing neuroprotection, there are still certain obstacles.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of pharmaceutical composition and its application containing withaferin A, described liquor-saturated
Eggplant element A and Quercetin are 1:(1~1500 in mass ratio) under can prevent and treat neurodegenerative disease, being not only substantially reduced respectively makes
With dosage, can more achieve the effect that better than exclusive use.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
The present invention provides a kind of pharmaceutical composition containing withaferin A, including withaferin A and Quercetin, the withaferin As
Mass ratio with Quercetin is 1:(1~1500).
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal in preparation prevention and treatment neurodegenerative disease
Application in drug.
Preferably, the neurodegenerative disease includes Parkinson's disease and/or Alzheimer disease.
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal in the drug for preparing neuro-protective
Using.
Preferably, by promoting the cell survival of nerve cell, neuro-protective is played.
Preferably, neuroprotective is played by anti-apoptotic.
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal to improve substantia nigra tyrosine hydroxylase in preparation
Application in the drug of positive neuron distribution and form.
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal in preparation treatment neuroinflamation drug
Using.
Preferably, the drug is using withaferin A and Quercetin as active constituent, further include pharmaceutically acceptable auxiliary material or
Complementary ingredient.
Preferably, the dosage form of the drug includes one of tablet, pulvis, granule, capsule, oral solution and sustained release agent
Or it is several.
The present invention provides a kind of pharmaceutical composition and its application containing withaferin A, the matter of the withaferin A and Quercetin
Amount is than being 1:(1~1500).The withaferin A and Quercetin are 1:(1~1500 in mass ratio) under can prevent and treat neurological
Property disease, neuron is protected, improve substantia nigra tyrosine hydroxylase positive neuron distribution and form, treat neuritis
Disease is not only substantially reduced respective dosage, can more achieve the effect that better than exclusive use.
Detailed description of the invention
Fig. 1 be MPTP induction Parkinson disease model group and control group in, respectively be injected intraperitoneally control solvent, withaferin A,
Quercetin and withaferin A+Quercetin (dosage halves) are after 7 days, the situation of change of mice behavior index；
Fig. 2 be MPTP induction Parkinson disease model group and control group in, respectively be injected intraperitoneally control solvent, withaferin A,
Quercetin and withaferin A+Quercetin (dosage halves) are after 7 days, mouse substantia nigra compacta tyrosine hydroxylase positive neurons
Form；
Fig. 3 be MPTP induction Parkinson disease model group and control group in, respectively be injected intraperitoneally control solvent, withaferin A,
Quercetin and withaferin A+Quercetin (dosage halves) are after 7 days, the variation of mouse striaturn tyrosine hydroxylase enzyme level；
Fig. 4 be MPTP induction Parkinson disease model group and control group in, respectively be injected intraperitoneally control solvent, withaferin A,
Quercetin and withaferin A+Quercetin (dosage halves) are after 7 days, the mouse substantia nigra compacta GFAP positive and Iba1 positive cell
It is horizontal；
Fig. 5 is that control solvent, withaferin A, Quercetin and withaferin A+Quercetin is added in the nerve cell of MPP+ damage
(dosage halves) cell survival rate afterwards；
Fig. 6 is that control solvent, withaferin A, Quercetin and withaferin A+Quercetin is added in the nerve cell of MPP+ damage
(dosage halves) Apoptosis situation afterwards；
Fig. 7 is H2O2Caused by oxidative damage nerve cell be added control solvent, withaferin A, Quercetin and liquor-saturated eggplant element
A+ Quercetin (dosage halves) cell survival rate afterwards.
Specific embodiment
The present invention provides a kind of pharmaceutical composition containing withaferin A, the mass ratio of the withaferin A and Quercetin is 1:
(1~1500).
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal in preparation prevention and treatment neurodegenerative disease
Application in drug.In the present invention, the drug further includes pharmaceutically preferably using withaferin A and Quercetin as active constituent
Acceptable auxiliary material or complementary ingredient.In the present invention, the dosage form of the drug preferably includes tablet, pulvis, granule, glue
One or more of capsule, oral solution and sustained release agent.Dosage of the present invention to the withaferin A and Quercetin in above-mentioned dosage form
It is not particularly limited, using the dosage of drug in regular dosage form.Auxiliary material that the present invention uses the above-mentioned dosage form or
The type and content of complementary ingredient are not particularly limited, using routine.
The present invention is not particularly limited the source of the withaferin A and Quercetin, using conventional commercial product.
In the present invention, the neurodegenerative disease preferably includes Parkinson's disease and/or Alzheimer disease.
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal in the drug for preparing neuro-protective
Using.In the present invention, by promoting the cell survival of nerve cell, neuro-protective is played；Or it is played by anti-apoptotic
Neuro-protective.In the present invention, the drug further includes pharmaceutically may be used preferably using withaferin A and Quercetin as active constituent
The auxiliary material of receiving or complementary ingredient.In the present invention, the dosage form of the drug preferably includes tablet, pulvis, granule, glue
One or more of capsule, oral solution and sustained release agent.Dosage of the present invention to the withaferin A and Quercetin in above-mentioned dosage form
It is not particularly limited, using the dosage of drug in regular dosage form.Auxiliary material that the present invention uses the above-mentioned dosage form or
The type and content of complementary ingredient are not particularly limited, using routine.
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal to improve substantia nigra tyrosine hydroxylase in preparation
Application in the drug of positive neuron distribution and form.In the present invention, the drug is preferably with withaferin A and Quercetin
Active constituent further includes pharmaceutically acceptable auxiliary material or complementary ingredient.In the present invention, the dosage form of the drug is preferably wrapped
Include one or more of tablet, pulvis, granule, capsule, oral solution and sustained release agent.The present invention is to the withaferin A and Mongolian oak
Dosage of the Pi Su in above-mentioned dosage form is not particularly limited, using the dosage of drug in regular dosage form.The present invention is to described
The type and content of auxiliary material or complementary ingredient that above-mentioned dosage form uses are not particularly limited, using routine.
The present invention also provides the pharmaceutical compositions described in above-mentioned technical proposal in preparation treatment neuroinflamation drug
Using.In the present invention, the drug further includes pharmaceutically acceptable preferably using withaferin A and Quercetin as active constituent
Auxiliary material or complementary ingredient.In the present invention, the dosage form of the drug preferably includes tablet, pulvis, granule, capsule, takes orally
One or more of liquid and sustained release agent.The present invention is to the no spy of the dosage of the withaferin A and Quercetin in above-mentioned dosage form
It is different to limit, using the dosage of drug in regular dosage form.The auxiliary material or complementary that the present invention uses the above-mentioned dosage form
The type and content of ingredient are not particularly limited, using routine.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Mouse used in following embodiment are as follows: SPF grades of C57 BL/6 mouse of 7-8 week old male are bought in Peking University
Medical board Laboratory Animal Science portion.C57 BL/6 mouse rearing conditions: 22 ± 0.5 DEG C of environment temperature, 12 hours/12 hours light and shades
Alternately.Mean ± standard error the expression of all experimental datas, * P < 0.05, * * P < 0.01, * * * P < 0.001, n=10.
Embodiment 1
Treat Parkinson's disease:
(corelation behaviour index)
MPTP induces Parkinson disease model to establish: after mouse adaptive feeding 1 week, randomly choosing the above C57 of weight 22g
BL/6 mouse, intraperitoneal injection 30mg/kg MPTP solution, 1 day 1 time, continuous 5 days.
At this point, MPTP injection group (hereinafter model group) is randomly divided into 4 groups, i.e. model+control solvent group, model+
Withaferin A group, model+Quercetin group, model+withaferin A+Quercetin group.Wherein, liquor-saturated eggplant element is injected intraperitoneally in every group of mouse respectively
A (20 μ g/kg), Quercetin (30mg/kg), withaferin A+Quercetin drug combination (withaferin A 10 μ g/kg, Quercetin 15mg/
) and isometric control solvent kg.
It detects after every group of drug and control solvent be injected intraperitoneally 7 days, the situation of change of each group behavioral indexes.
Behavioral indexes detection: after the Parkinson disease model of MPTP induction is established, in the 4th day (materials of each group drug injection
First 3 days) start to carry out, in quiet, suitable environment, the practice of mice behavior Indexs measure is carried out, each tests every daily test
It surveys 3 times, to exclude interference of the other factors to behavioral indexes.
1) Beam balance test
1. mouse is moved to test room, habituation 1h.
2. prepare one group of long 100cm, diameter be respectively 10mm, 20mm, 30mm round bar and it is wide be respectively 5mm, 15mm,
The square bar of 30mm.
3. constructing the platform of two liftoff about 50cm, two platforms are at a distance of about 80cm, wherein a platform places a darkroom.It will
Above-mentioned most wide bar is taken between two platforms and fixed.
4. mouse to be headfirst placed in the platform of darkroom opposite side, with Qiang Guang or noise stimulation its Xiang Qianhang behind it
It walks, records it and arrive at darkroom required time and period hind leg skidding number.
5. wide bar is replaced with narrow bar, testing procedure is similarly.
6. being tested daily 5 times with a batch mouse, follow-on test three days, it is averaged, the results are shown in Table 1 and Fig. 1.
1 Beam balance test result of table
2) Grasping clubglass test
1. mouse is moved to test room, habituation 1h.
2. preparing a root long 50-60cm, diameter is the round bar of 1-1.5cm, anti-skidding with gauze package, and it is vertically stood up
It sets.
3. mouse to be head-up placed in round bar top, round bar bottom is recorded the time required to it switchs to upside down and reached
Required time.
4. being tested daily 5 times with a batch mouse, follow-on test three days, it is averaged, the results are shown in Table 2 and Fig. 1.
2 Grasping clubglass test result of table
3) hind leg holds experiment
1. mouse is moved to test room, habituation 1h.
2. position is lifted among crawl mousetail, with its hind leg active state of camera record, 20s is recorded altogether.
3. being scored according to mouse hind leg activity condition: in 0 point-test process, hind leg is in nature open configuration；1 point-
Side hind leg occurs holding or two sides hind leg is slightly held；2 points-two sides hind leg the most of the time holds, but still has flexibility；
3 points-two sides hind leg is held completely, no flexibility.
4. being tested daily 3 times with a batch mouse, follow-on test three days, it is averaged, the results are shown in Table 3 and Fig. 1.
3 hind leg of table holds experimental result
4) rotary bar is tested
1. mouse is moved to test room, habituation 1h.
2. mouse is placed on test equipment (bull stick), initial velocity 4rpm is ramped up, and every 8s increases 1rpm,
In 5min, revolving speed is increased to after 40rpm and is not further accelerated.
3. the time that record mouse falls from bar
4. testing daily 3 times with a batch mouse, every minor tick 20min follow-on test three days, is averaged, the results are shown in Table
4。
Wherein, compared with normal group, model group behavioral indexes are significantly lowered, and apparent dyskinesia occur；And liquor-saturated eggplant
Plain A+ Quercetin drug combination group can significantly restore mouse movement dysfunction caused by MPTP, and effect is given better than single
Medicine group (the result is shown in Figure 1).
4 rotary bar experimental result of table
Embodiment 2
Establish the MPTP-induced Parkinson disease mice model of MPTP induction (method is with embodiment 1).By MPTP injection group (hereinafter mould
Type group) be randomly divided into 4 groups, i.e. model+control solvent group, model+withaferin A group, model+Quercetin group, model+withaferin A+
Quercetin group.Wherein, withaferin A (20 μ g/kg), Quercetin (3mg/kg), withaferin A+Mongolian oak is injected intraperitoneally in every group of mouse respectively
Skin element drug combination (withaferin A 10 μ g/kg, Quercetin 1.5mg/kg) and isometric control solvent.
It detects after every group of drug and control solvent be injected intraperitoneally 7 days, the situation of change of each group behavioral indexes.Balance beam is real
The method tested the results are shown in Table 5 with embodiment 1.
5 Beam balance test result of table
Grasping clubglass test method the results are shown in Table 6 with embodiment 1.
6 Grasping clubglass test result of table
Hind leg holds experimental method with embodiment 1, the results are shown in Table 7.
7 hind leg of table holds experimental result
Rotary bar experimental method the results are shown in Table 8 with embodiment 1.
8 rotary bar experimental result of table
The result shows that withaferin A+Quercetin drug combination (withaferin A 10 μ g/kg, Quercetin 1.5mg/kg) can be shown
It writes and restores mouse movement dysfunction caused by MPTP, and effect is better than single administration group (the results are shown in Table 5-6).
Embodiment 3
Protective effect to black substance and striatal dopaminergic neuron:
Tyrosine hydroxylase (TH) is the rate-limiting enzyme of intracerebral dopamine synthesis, horizontal height, represents mouse synthesis DOPA
The ability of amine, therefore, often using tyrosine hydroxylase as the index of mouse dopamine secretion ability.
After MPTP model foundation, the foundation of MPTP model and the dosage of drug detect every group of drug and control with embodiment 1
After solvent is injected intraperitoneally 7 days, using immunohistochemistry technique each group mouse substantia nigra compacta tyrosine hydroxylase enzyme level, it the results are shown in Table
9。
As a result are as follows: compared with normal group, model group mouse substantia nigra compacta TH positive neuron quantity is remarkably decreased, that is, is gone out
Now apparent dopaminergic apoptotic neurons situation.And withaferin A+Quercetin drug combination group can significantly restore MPTP and be drawn
The dopaminergic apoptotic neurons risen, and effect is better than single administration group (result is shown in Fig. 2).
9 tyrosine hydroxylase of table (TH) positive neuron quantity statistics
In addition, dopaminergic neuron projection significantly reduces in model group mouse striaturn, and withaferin A+Quercetin connection
This case can significantly be restored by sharing medicine, and effect is better than single administration group (Fig. 3).
Embodiment 4
Treat neuroinflamation:
It is now recognized that the generation of neuroinflamation and development are one of the correlative factor of neurodegenerative disease morbidity, nerve
Inflammation promotes the effect of mediate neuronal progressive death to be confirmed extensively in neurodegenerative disease process.Neuroinflamation
Generation mainly include the inflammatory reactions such as microglial activation, Activation of Astrocytes.The inflammatory of these further maincenters
Cell is interacted by number of mechanisms, is generated proinflammatory cytokine and is amplified inflammatory signals, generation acts directly on neuron
Neurotoxin finally results in the generation of neurodegenerative disease.
After MPTP model foundation, the foundation of MPTP model and the use of drug detect every group of drug and control with embodiment 1
After solvent is injected intraperitoneally 7 days, immunohistochemistry technique each group mouse substantia nigra compacta GFAP and Iba1 positive cell is utilized.Wherein,
GFAP and Iba1 is respectively the marker of astroglia and microglia, and abnormal activation indicates neuroinflamation level
Rise, the results are shown in Table 10.
The result shows that withaferin A+Quercetin drug combination group can significantly reduce neuroinflamation level, and effect is better than
Single administration group (result is shown in Fig. 4).
The 10 each group mouse black substance GFAP positive of table and Iba1 positive cell quantity statistics
Embodiment 5
Neuroprotection-MTT experiment:
SH-SY5Y cell is mankind's neuroma mother cell cell line, is usually used in the experiment in vitro of neurodegenerative disease.To
After 50 μM of MPP+ of toxicant are added in SH-SY5Y cell, there is significant neurotrosis situation.At this point, adding respectively into cell
Enter withaferin A (0.1 μM), Quercetin (200 μM), control solvent (DMSO) and withaferin A+Quercetin joint group (0.1 μM+
100μM)。
MTT, that is, Methylthiazolyldiphenyl-tetrazoliumbromide, also referred to as Thiazolyl blue
Tetrazoliumbromide, the entitled thiazolyl blue of Chinese.Molecular formula is C18H16BrN5S, molecular weight 414.32.It detects former
Manage the bluish violet Jie Jing formazan that exogenous MTT can be made to be reduced to water-insoluble for the succinate dehydrogenase in living cells mitochondria
(Formazan) it and is deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) can dissolve formazan in cell,
Its absorbance value is measured with microplate reader.Within the scope of certain cell number, it is directly proportional to cell number that MTT crystallizes the amount to be formed.According to
The absorbance value (OD value) measured, to judge living cells quantity, OD value is bigger, and cell activity is stronger, that is, can determine whether drug to mind
Protective effect through cell.
1) MTT powder (500mg) is configured to 5mg/ml and is dissolved in sterile PBS solution that (500mgMTT powder, is dissolved in
In the sterile PBS of 100ml, two 50ml centrifuge tubes), after mixing completely, after 0.22 μm of membrane filtration, it is loaded in EP pipe, be protected from light-
20 DEG C of long-term preservations.
2) pancreatin digests logarithmic phase cell, is collected by centrifugation after termination, cell suspension is made, cell count adjusts its concentration extremely
5-10×104/ml。
3) it after preparing cell suspension, mixes gently, 100 μ l are added in every hole, and the density of cell to be measured in this way is 5000-
10000/ hole (edge hole is filled with sterile PBS).
4) inoculated tissue culture plate is put into incubator and is cultivated, (96 holes are flat until cell monolayer is paved with bottom hole
Plate), the drug of concentration gradient is added, after cell is adherent can dosing, about 6h or so, every 100 μ l of hole, if 3-6 multiple holes.
4-1) drug is added in 96 orifice plates by different volumes, to form concentration gradient.
4-2) drug of various concentration is prepared in EP pipe, then removing the culture supernatant in 96 orifice plates (can use
The volley of rifle fire siphons away) add the culture mediums of 100 μ l drugs containing various concentration.
5) 5%CO2, 37 DEG C incubation 16-48 hours, under inverted microscope observe drug function and effect.
6) 10 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) are added in every hole, continue to cultivate 4h.If drug and MTT can
Reaction, can first be centrifuged and discard culture solution afterwards, carefully rush 2-3 after with PBS, add the culture solution containing MTT.
7.) culture is terminated, is prepared dissolving crystallized.
After 7-1) culture 4h is added in MTT, crystallization can be sufficiently formed.Supernatant is removed, the process is it is noted that cannot be crystallization
It removes.
7-2) 150 μ l dimethyl sulfoxides are added in every hole, set low-speed oscillation 10min on shaking table, dissolve crystal sufficiently.In
The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm.
According to the absorbance value (OD value) measured, to judge living cells quantity, OD value is bigger, and the stronger result of cell activity is shown in
Table 11.
As a result are as follows: withaferin A+Quercetin drug combination can be obviously improved the cell survival rate of nerve cell, play mind
Through first protective effect (result is shown in Fig. 5).
11 group of cells survival rate (%) of table
Embodiment 6
Anti-apoptotic:
Fluorescent dye PI (propidium iodide) is a kind of nuclei dyeing color reagent that can be dyed to DNA, is usually used in Apoptosis
Detection, full name in English is Propidium Iodide.It is a kind of analog of Ethidum Eremide, is discharged after being embedded in double-stranded DNA
Red fluorescence.Although PI cannot be by living cells film, can be across damaged cell membrane and to nuclear staining.
After 50 μM of MPP+ of toxicant are added to SH-SY5Y cell, withaferin A (0.1 μM) is added into cell respectively,
Quercetin (200 μM), control solvent (DMSO) and withaferin A+Quercetin joint group (0.1 μM+100 μM).
5%CO2, after 37 DEG C of incubations are cultivated 24 hours, PBS is washed 1-2 times, and ofpropidium iodide solution (1mg/ml) and 7uM is added
Hochest (label nucleus) is incubated for 15 minutes.Dyeing liquor is discarded, PBS is washed 1-2 times, mounting.Fluorescence microscope shooting, calculates
Propidium iodide red fluorescence and Hochest blue-fluorescence label cell ratio the results are shown in Table to get Apoptosis situation data
12。
12 group of cells apoptosis situation (%) of table
The experimental results showed that withaferin A+Quercetin drug combination group, can play neuron significantly to anti-apoptotic
As a result Fig. 6 is shown in protective effect.
Embodiment 7
It is anti-oxidant, neuroprotection:
100 μM of H of toxicant are added to SH-SY5Y cell2O2Afterwards, there is significant neurotrosis situation.At this point, respectively
Addition withaferin A (1 μM), Quercetin (20 μM), control solvent (DMSO) and withaferin A+Quercetin joint group into cell
(0.5 μM+10 μM), the results are shown in Table 13.
As a result are as follows: withaferin A+Quercetin drug combination can be obviously improved the cell survival rate of nerve cell, play mind
Through first protective effect (result is shown in Fig. 7).
13 group of cells survival rate (%) of table
Above embodiments show that withaferin A joint Quercetin can be substantially reduced respective dosage, remain to reach and are better than
The effect of exclusive use is that the use in conjunction with huge clinical landscapes is found.
Wherein, oxidative stress and apoptosis are comprising the neurodegenerative disease including Alzheimer disease and Parkinson's disease
Main pathogenesis, and a kind of pharmaceutical composition provided by the invention (withaferin A and Quercetin combination), can be effective against oxygen
Change stress and apoptosis, protect neuronal activity, have the effect of anti-Alzheimer disease and Parkinson's disease.
Embodiment 8
Protective effect to the SH-SY5Y cellular damage of beta-amyloid protein induction
Amyloid beta abnormal accumulation in Alzheimer brain tissue, and then Ahl tribulus sea silent sickness phase of setting out
The pathologic, physiologic of pass, biochemical relevant cascade reaction.Therefore, the toxic effect of beta-amyloid protein is fought, can play and be directed to
The preventive and therapeutic action of Alzheimer disease.
SH-SY5Y presses cell density 1 × 105It is inoculated in porous plate, after cell is adherent, is added to SH-SY5Y cell
After 15 μM of beta-amyloid proteins, withaferin A (0.1 μM) is added into cell respectively, Quercetin (200 μM), control solvent
(DMSO) and withaferin A+Quercetin joint group (0.1 μM+100 μM), culture are tested afterwards for 24 hours.
1) MTT powder (500mg) is configured to 5mg/ml and is dissolved in sterile PBS solution that (500mgMTT powder, is dissolved in
In the sterile PBS of 100ml, two 50ml centrifuge tubes), after mixing completely, after 0.22 μm of membrane filtration, it is loaded in EP pipe, be protected from light-
20 DEG C of long-term preservations.
2) pancreatin digests logarithmic phase cell, is collected by centrifugation after termination, cell suspension is made, cell count adjusts its concentration extremely
5-10×104/ml。
3) it after preparing cell suspension, mixes gently, 100 μ l are added in every hole, and the density of cell to be measured in this way is 5000-
10000/ hole (edge hole is filled with sterile PBS).
4) inoculated tissue culture plate is put into incubator and is cultivated, (96 holes are flat until cell monolayer is paved with bottom hole
Plate), the drug of concentration gradient is added, after cell is adherent can dosing, about 6h or so, every 100 μ l of hole, if 3-6 multiple holes.
4-1) drug is added in 96 orifice plates by different volumes, to form concentration gradient.
4-2) drug of various concentration is prepared in EP pipe, then removing the culture supernatant in 96 orifice plates (can use
The volley of rifle fire siphons away) add the culture mediums of 100 μ l drugs containing various concentration.
5) 5%CO2, 37 DEG C incubation 16-48 hours, under inverted microscope observe drug function and effect.
6) 10 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) are added in every hole, continue to cultivate 4h.If drug and MTT can
Reaction, can first be centrifuged and discard culture solution afterwards, carefully rush 2-3 after with PBS, add the culture solution containing MTT.
7.) culture is terminated, is prepared dissolving crystallized.
After 7-1) culture 4h is added in MTT, crystallization can be sufficiently formed.Supernatant is removed, the process is it is noted that cannot be crystallization
It removes.
7-2) 150 μ l dimethyl sulfoxides are added in every hole, set low-speed oscillation 10min on shaking table, dissolve crystal sufficiently.In
The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm.
According to the absorbance value (OD value) measured, to judge living cells quantity, OD value is bigger, and the stronger result of cell activity is shown in
Table 14.
14 group of cells survival rate (%) of table
Thus after table can be seen that addition beta-amyloid protein, cell can be significantly killed, cell survival rate drop to normally
The 33% of group, and withaferin A and Quercetin administering drug combinations group can significantly fight beta-amyloid protein toxicity, protect neuron,
And effect is better than single administration group.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of pharmaceutical composition containing withaferin A, which is characterized in that including withaferin A and Quercetin, the withaferin A with
The mass ratio of Quercetin is 1:(1~1500).
2. application of the pharmaceutical composition described in claim 1 in the drug of preparation prevention and treatment neurodegenerative disease.
3. application according to claim 2, which is characterized in that the neurodegenerative disease include Parkinson's disease and/or
Alzheimer disease.
4. application of the pharmaceutical composition described in claim 1 in the drug for preparing neuro-protective.
5. application according to claim 4, which is characterized in that by promoting the cell survival of nerve cell, play nerve
Member protection.
6. application according to claim 4, which is characterized in that play neuroprotective by anti-apoptotic.
7. pharmaceutical composition described in claim 1 improves the distribution of substantia nigra tyrosine hydroxylase positive neuron and form in preparation
Drug in application.
8. application of the pharmaceutical composition described in claim 1 in preparation treatment neuroinflamation drug.
9. described in any item applications according to claim 1~8, which is characterized in that the drug is with withaferin A and Quercetin
Active constituent further includes pharmaceutically acceptable auxiliary material or complementary ingredient.
10. application according to claim 9, which is characterized in that the dosage form of the drug include tablet, pulvis, granule,
One or more of capsule, oral solution and sustained release agent.
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