CN109288852A - The purposes of Quzhazhigan or derivatives thereof - Google Patents
The purposes of Quzhazhigan or derivatives thereof Download PDFInfo
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- CN109288852A CN109288852A CN201811382448.9A CN201811382448A CN109288852A CN 109288852 A CN109288852 A CN 109288852A CN 201811382448 A CN201811382448 A CN 201811382448A CN 109288852 A CN109288852 A CN 109288852A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
Abstract
The present invention relates to the purposes of biopharmaceutical technology more particularly to Quzhazhigan or derivatives thereof.The present invention research shows that; Quzhazhigan or derivatives thereof, which has, to be inhibited toxicity of excitatory amino acid, anti-nerve cell apoptosis, improves neural cell activity, reduces MDA level in nerve cell; the effects of promoting neurocyte proliferation, and there are significant protective effects to ALS cell model for Quzhazhigan or derivatives thereof.
Description
Technical field
The present invention relates to the purposes of biopharmaceutical technology more particularly to Quzhazhigan or derivatives thereof.
Background technique
Motor neuron disease (MND) is the unknown selection assault sexually spinal cord anterior horn cell of one group of cause of disease, brain stem kinesitherapy nerve
The chronic progressive neurodegenerative disease of member, cortex pyramidal cell and pyramidal tract.According to the difference of clinical manifestation, motor neuron
Disease can be generally divided into following four type: amyotrophic lateral sclerosis (ALS)；Progressive muscular atrophy (PMA)；Progressive prolongs
Marrow benumbs (PBP)；Primary lateral sclerosis (PLS).Its harmfulness is that selectivity is driven in the wrong direction and damages motor neuron, shows as mind
Through first limited fine-motoric dominated, atrophy, such as involve bottleneck throat, respiratory muscle will lead to life termination.Furthermore involve movement
Neuron cause dominated muscle activity cannot or it is uncoordinated, cause to be easy wrestling even life danger occur, to patient and
Its family life quality, psychosoma and economic level all bring serious injury and influence.ALS is a kind of selectivity that the cause of disease is unknown
The chronic progressive degenerative disease of spinal cord anterior horn cell, brain stem kinesitherapy nerve core and pyramidal tract is invaded, clinical manifestation is to transport up and down
Dynamic neuron merges impaired sign, is the most common type in chronic exercise neuronal disease.Its symptom has muscular strength decline, flesh
Meat atrophy, and gradually development is that diet is choked cough, dysphagia, expiratory dyspnea, until death, 90% ALS is sporadic
(SALS), 10% ALS is familial (FALS).
The pathogenesis of ALS is still not clear, and may relate to the missing of toxicity of excitatory amino acid effect, neurotrophic factor
It is reacted with exception, oxidative stress, protein Misfolding and abnormal aggregation, mitochondria dysfunction, apoptosis, neuritic, is non-
Motor neuron participation etc..The effect of inflammation and oxidative stress in the motor nerve degeneration that SOD1 is mediated becomes recent
Research hotspot.
The disease lacks effective treatment method at present, and the survival period of patient is generally 3-5.Although there are many medicines for many years
Object is studied, Riluzole (riluzole tablets, its chemical name is: trifluoromethoxybenzathiazole) still
The only drug for being applied to treatment ALS by FDA approval, but its also can only the limited life cycle for extending patient, and the medicine
Extremely expensive, most patient homes are difficult to bear, which also results in liver and kidney dysfunction, therefore are badly in need of exploitation and more pacify
Entirely, economically and efficiently drug.
In recent years, scientists are according to possible pathogenesis such as toxicity of excitatory amino acid effect, oxidative stress and line
Mitochondria function obstacle, apoptosis, immune and Neuroinflammation, the missing of neurotrophic factor and exception, gene mutation, environment because
The hypothesis such as element have conducted extensive research, it is intended to search out effective treatment method.Quzhazhigan ((E) -1- (3,5- dihydroxyphenyls
Base) -2- (3- hydroxyl -4-O- β-D- glucopyranose phenyl) ethylene or 3,5,3', 4'- tetrahydroxy Stilbene -3'-O- β-glucose
Glycosides), plant origin is Lhasa rhubarb rhizome, and piceatannol is the aglycon of Quzhazhigan.Chinese patent (application number:
201010116358.2) " application and preparation method thereof of the Quzhazhigan in preparation prevention and treatment cardiac-cerebral ischemia basic preparation "；China
A kind of patent (application number: 2011110371198.0) " high performance liquid chromatography side for measuring Quzhazhigan content in Lhasa rhubarb
Method "；Chinese patent (application number: 201110253242.8) " a method of prepare piceatannol " disclose mentioning for Quzhazhigan
Taking technique, detection method and prepare Quzhazhigan aglycon piceatannol method.
The prior art indicate that Quzhazhigan and its derivative have the activity in terms for the treatment of ischemic angiocardiopathy and cerebrovascular disease, but
It has no and is treating and preventing the purposes report in ALS drug.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that provide the invention discloses Quzhazhigans or derivatives thereof
Medical usage, the present invention be found through experiments that Quzhazhigan or derivatives thereof have inhibit toxicity of excitatory amino acid, improve
The effects of neural cell activity reduces MDA level in nerve cell, promotes neurocyte proliferation, and Quzhazhigan or its derivative
There are significant protective effects to ALS cell model for object.
The present invention provides application of the Quzhazhigan or derivatives thereof in the preparation of preparation protection nerve cell.
In the embodiment of the present invention, the protection is horizontal to reduce MDA in nerve cell.The nerve cell is that neuron is thin
Born of the same parents；In some embodiments, the neuronal cell is normal neuronal cell, in other embodiments, the neuronal cell
To transfect hSODlG93ANeuronal cell.The normal neuronal cell is NSC34 cell or transfection hSODlWTPlasmid
NSC34 cell.
In this embodiment, it is transferred to hSODl into the cell in NSC34G93AAnd hSODlWTPlasmid, with establish ALS cell model and
Control cell model.The ALS disease cells model building method for being configured to this field approval of this model.In the NSC34 of transfection
Give 10,20,40 μm of olL respectively into the cell-1Quzhazhigan, after piceatannol or resveratrol culture 48h, find cell
Form is without significant change.Compared with the NSC34 cell normally cultivated, hSODlG93AMDA content obviously rises in NSC34 cell
High (P < 0.001), and giving 20,40 μm of olL-1Quzhazhigan, piceatannol administration after, MDA content be substantially reduced (P <
0.01), and certain dosage correlation is presented；And resveratrol is to the reduction no difference of science of statistics of MDA content,
In the embodiment of the present invention, the protection is raising nerve cell vigor.The nerve cell is neuronal cell；One
In a little embodiments, the neuronal cell is normal neuronal cell, and in other embodiments, the neuronal cell is transfection
hSODlG93ANeuronal cell.The normal neuronal cell is NSC34 cell or transfection hSODlWTThe NSC34 of plasmid is thin
Born of the same parents.
In this embodiment, 10,20,40 μm of olL are given respectively into the cell in the NSC34 of transfection-1Quzhazhigan, white skin
After China fir alcohol or resveratrol culture 48h, 20,40 μm of olL are being given-1Cell number after Quzhazhigan, piceatannol intervention
It increased (P < 0.05) compared with the cell number of non-administered group；Resveratrol has increase trend, but no difference of science of statistics.
In the embodiment of the present invention, the protection is to inhibit neural information encoding, the apoptosis for inhibiting nerve cell and/or promotion
The proliferation of nerve cell.
In some embodiments, the neural information encoding is the neural information encoding of glutamate induction.
The present invention experiments have shown that, glutamic acid causes nerve cell (human neuroblastoma cells) under 100mM dosage
Exitotoxicity damage, for the SH-SY5Y cellular neural exitotoxicity of glutamate induction, Quzhazhigan and piceatannol are in 300 μ
Neurotoxic effect can be significantly fought under M dosage, resveratrol effect is unobvious.
In the present invention, compared with sham-operation group, model group rats cerebral ischemia-fill again after for 24 hours, intracerebral TUNEL method dyeing mark
The apoptotic cell and Cleaved Caspase-3 immuno positive (Cleaved Caspase-3-ir) cell of note increase
(P < 0.01), Quzhazhigan 4mg/kg group can significantly reduce intracerebral TUNEL method dye marker apoptotic cell and
Cleaved Caspase-3 immuno positive (Cleaved Caspase-3-ir) cell
In other embodiments, compared with sham-operation group, model group animal brain transient ischemia/reperfusion for 24 hours after, the water of intracerebral Ki67
Head up display work increases (P < 0.01), the trend that Ki67 is reduced after Quzhazhigan 4mg/kg administration, but there was no significant difference (with model
Group compares, P > 0.05).While transient ischemia/reperfusion, BrdU is injected intraperitoneally, then after filling for 24 hours, brain area can be observed in ischemic side
Level to BrdU significantly increases (P < 0.01vs sham-operation group), and this effect can be given Quzhazhigan 4mg/kg institute by vein
It fights (P < 0.01vs model group).BrdU is given within continuous two weeks, the level of model group animal ischemic side brain area BrdU then significantly subtracts
It is few, and the level for giving Quzhazhigan group rat ischemia side brain area BrdU then increased significantly (P < 0.05vs model group).This knot
Fruit explanation, Quzhazhigan have the function of promoting cell Proliferation.
Studies have shown that Quzhazhigan or derivatives thereof has the function of antioxidation in vitro.
Experiment shows piceatannol and Edaravone to O2 ?Scavenging activity it is close, IC50Respectively 5.81,13.85 μ
M；Quzhazhigan, resveratrol have certain scavenging effect, and Vc fails to measure IC50.Piceatannol is most strong to OH Scavenging activity,
IC50It is 9.29 μM；Vc, Quzhazhigan take second place, and resveratrol is most weak, IC50Respectively 27.2 μM, 36.82 μM, 50.24 μM.It is bent
Letter Stilbene glycosides is to ABTS.+With stronger Scavenging activity, IC50It is 4.28 μM；Resveratrol, piceatannol and Vc take second place, IC50Respectively
It is 4.47,11.12 and 15.52 μM.Piceatannol reducing power is most strong, EC50It is 38.22 μM；Quzhazhigan and Vc, Yi Dala
Give effect quite, resveratrol is most weak, EC50Respectively 127.1,80.18,94.55,136.09 μM.
Based on result above, the present invention provides Quzhazhigans or derivatives thereof in the medicine for preparing prevention and treatment motor neuron disease
It is applied in object.Specifically, the motor neuron disease is amyotrophic lateral sclerosis.
Quzhazhigan and its derivative, which have, to be inhibited toxicity of excitatory amino acid, improves neural cell activity, reduces nerve
MDA is horizontal in cell, promotes the neuroprotections such as neurocyte proliferation, according to the possible pathogenesis of ALS, Quzhazhigan and
Its derivative can be intervened for the above pathogenesis, play preventive and therapeutic action to ALS.
In the present invention, the derivative of the Quzhazhigan is piceatannol.
The present invention also provides a kind of drugs for preventing and treating motor neuron disease comprising in Quzhazhigan or derivatives thereof
It is at least one.
The dosage form of drug of the present invention is tablet, capsule, oral solution, suspension, injection etc..
The present invention also provides a kind of methods for preventing and treating motor neuron disease, to give drug of the present invention.Institute
Stating motor neuron disease is amyotrophic lateral sclerosis.The dosage given is 4mg/kg.
The present invention inhibits toxicity of excitatory amino acid, anti-nerve cell studies have shown that Quzhazhigan or derivatives thereof has
The effects of apoptosis improves neural cell activity, reduces MDA level in nerve cell, promotion neurocyte proliferation, and Quzhazhigan
Or derivatives thereof to ALS cell model, there are significant protective effects.Experimental result is shown: (1) Quzhazhigan, piceatannol energy
It is obviously promoted transfection hSODlG93ANSC34 cell survival after plasmid, vigor obviously increase (P < 0.001)；And there is reduction MDA
Effect (P < 0.01).(2) Quzhazhigan is most strong to the scavenging effect of ABTS+ in vitro, and piceatannol takes second place；It is removing
O2 ?, piceatannol effect is most strong in terms of OH and total reducing power, Quzhazhigan takes second place.(3) for the mind of glutamate induction
Through exitotoxicity, Quzhazhigan and piceatannol can significantly fight neurotoxic effect under 300 μM of dosage.(4) bent letter Stilbene
Glycosides 4mg/kg can significantly reduce line brush induction rat focal cerebral ischemia in TUNEL method dye marker apoptotic cell with
And Cleaved Caspase-3 immuno positive (CleavedCaspase-3-ir) cell, prompt Quzhazhigan 4mg/kg to have anti-
Neuron Apoptosis effect；Chronic cerebral ischemia in rats ischemic side brain area BrdU level can be made to increased significantly simultaneously, prompt Quzhazhigan
There is obvious repair to nerve cell.
Detailed description of the invention
Fig. 1 shows influence of the Quzhazhigan to NSC34 cell viability；
Fig. 2 shows Quzhazhigan to hSODlG93AAnd hSODlWTThe influence of NSC34 cell viability；
Fig. 3 shows Quzhazhigan to hSODlG93AAnd hSODlWTThe influence of MDA in NSC34 cell；
Fig. 4 shows FeSO4Relationship between reaction density and absorbance value.
Specific embodiment
The present invention provides the purposes of Quzhazhigan and its derivative, those skilled in the art can use for reference present disclosure,
It is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications carry out those skilled in the art
Say it is it will be apparent that they are considered as being included in the present invention.Method and application of the invention has passed through preferred embodiment
Be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to methods herein and application into
Row change or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The test material that the present invention uses is all common commercially available product, can all be bought in market.
Below with reference to embodiment, the present invention is further explained:
Effect of the embodiment 1 to amyotrophic lateral sclerosis cell model
1 test sample
Quzhazhigan, molecular weight 406, white crystals or crystalline powder, purity 99.6%, lot number 20120402；
Piceatannol, molecular weight 244, red-brown powder, purity 99.8%, lot number 906099a；
Resveratrol, molecular weight 228, white powder, purity 99.8%, lot number CY120325.
2 experimental cells and plasmid
NSC34 mouse neuronal cell is purchased from Guangzhou Ji Niou Biotechnology Co., Ltd.Cell culture, which is selected, contains 10%
The DMEM culture medium of FBS, is placed in 37 DEG C, 5%CO2Incubator in culture.PF141pAcGFPl SOD1WT and
PF145pAcGFPl SOD1G93A plasmid is by general as spit of fland biotechnology (Beijing) Co., Ltd constructs and is sequenced.
3 reagents and instrument
DMEM culture medium, poly-D-lysine, DMSO (Gibco, USA)；Lipofectamine2000 (Invtrogen,
USA)；Fetal calf serum (FBS, Hycolne, USA)；Penicillin-streptomysin (Sigma)；Ammonium persulfate, TEMED, thiazolyl blue
(MTT) (Sigma, USA)；Acrylamide, N', a N ' methylene-bisacrylamide (Amersco, USA)；SDS (Biomol,
USA)；Pvdf membrane (Millipore, USA)；BCA egg self-quantitatively kit (the green skies)；LipofectamineTM 2000
(invitrogen company)；5 × Loading Buffer (Fermentas, USA)；(bioengineering is built up in Nanjing to MDA kit
Research institute)；Remaining various reagents is import or domestic analytically pure reagent.
Key instrument: 3121 type carbon dioxide incubator (Thermo, USA) of ThermoForma；The desk-top freezing of Biofuge
Centrifuge (Thermo, USA))；XSE type electronic analytical balance (plum Teller, Switzerland)；The ultrapure water system of Milli-QIntegral 5
It unites (Millipore, USA)；Ultra low temperature freezer (Thermo, USA)；Synergy2 microplate reader (BioTek, USA)；V-GES electrophoresis
Instrument (Wealtec Crop)；STS-3 type decolorization swinging table (Shanghai Qi Te Analytical Instrument Co., Ltd)；ChemiDoc-It Imager
Type chemiluminescence imaging analysis system (UVP).
4 test methods
4.1 cell culture: being removed from liquid nitrogen the NSC34 cell frozen, and the DMEM training of the serum containing 10%FBS is added
Feeding base was cultivated, every 2 days one subcultures of replacement.It is placed in containing 5%CO237 DEG C of incubator cultures.
4.2 cell transfectings: the day before transfection uses NSC34 cell containing 0.25%EDTA pancreatin digestive inoculation in 6 orifice plates
In, culture cell grows to 90% convergence degree, operates transfection hSODl with 2000 by specification of LipofectamineWTWith
hSODlG93AEach 10 μ g of plasmid is changed to normal incubation medium after transfecting 6h, continues culture and with G418 400mgL-1It carries out thin
Born of the same parents' screening.
4.3 pharmaceutical interventions: add people 5 × 10 in coated 96 orifice plate4A mL-1100 μ L of cell suspension, or wrapping
2 × 10 are added in the 6 orifice plates of quilt5A mL-1Cell suspension 2mL, cultivate 48h after carry out pharmaceutical intervention processing.In advance with containing
The DMEM culture medium of 10%FBS dilutes Quzhazhigan, piceatannol, resveratrol to 10,20,40 μm of olL-1.96 orifice plates add
100 μ L of people or 6 orifice plates add people 2mL drug containing (experimental group) or not the DMEM complete medium of drug containing (control group), are placed in cell training
Culture in case is supported, observes cellular morphology and vigor after 48h.
4.4MTT detection: after pharmaceutical intervention 48h, every hole adds the MTT (50mgmL of 10 μ L of people in 96 orifice plates-1), after 4h
37 DEG C of three liquid of people is added to be incubated overnight, microplate reader measures OD value under the conditions of 570nm, analyzes the vigor of cell.
4.5 western blots: supernatant culture medium is sucked out in cell to be processed, is washed with PBS and is exhausted afterwards twice, people's cell is added to split
Solution liquid is placed on ultrasonication on ice, in 40 DEG C, 12000rmin-1It is centrifuged 30min, takes supernatant.With BCA protein quantification reagent
Box by operating instruction carry out it is quantitative after, every group adjusts consistent to protein concentration, add 5 × LoadingBuffer of 1/4 volume of people,
5min is boiled after mixing makes albuminous degeneration, -70 DEG C of preservations.Using egg from SDS-PAGE western blot method prepare 10% separation
Glue, equivalent loading are added antibody after carrying out electrophoresis, transferring film, closing, are developed the color using ECL, observe the expression of intracellular SOD1 albumen.
4.6MDA kit detection: take out cell culture 6 orifice plates, washed twice with 4 DEG C of PBS, then plus people 1mL PBS be used in combination
Cell scraper under cell scraper, will be transferred in 1.5mL Ep pipe, 1000rmin-1, it is centrifuged 5min, after supernatant carefully is sucked out, then
Add 200 μ L cell pyrolysis liquid of people, ice-water bath homogenate, 2500rmin-1, it is centrifuged 10min, takes supernatant by MDA kit explanation
Detection.
4.7 statistical procedures methods experiment data are analyzed using Graphpad Prism7.0 software, data withIt indicates, conspicuousness is examined between carrying out group using one-way analysis of variance (ANOVA) and Newman-keuls post-hoc
It tests.
5 test results
The influence of 5.1 pairs of NSC34 cells
For probe into Quzhazhigan, piceatannol, resveratrol to the NSC34 cell normally cultivated with the presence or absence of promote survival and
Cytotoxic effect, experiment give NSC34 cell with 10,20,40 μm of olL-1Quzhazhigan, piceatannol, resveratrol
48h is cultivated, and using the cellular morphology and vigour changes of micro- sem observation and MTT detection NSC34.With the NSC34 normally cultivated
Cell is compared, and is administered between each group and control group in cellular morphology and vigor no significant difference (P > 0.05), is seen Fig. 1.As a result table
Bright, in the NSC34 cell normally cultivated, Quzhazhigan, piceatannol are without obviously promoting survival or cytotoxic effect.
5.2 establish ALS cell model
This experiment is transferred to hSODl in NSC34 into the cellG93AAnd plasmid, to establish ALS experiment and control cell model.Knot
Fruit shows, in transfection hSODlG93AAnd hSODlWTAfter plasmid, the NSC34 and hSODl normally cultivated is examinedG93ANSC34 is intracellular
SOD1 protein expression level, further to verify hSODlG93AThe intracellular SOD1 protein expression level of NSC34.The result shows that
hSODlG93AThe intracellular SOD1 level of NSC34 obviously increases, the success of ALS model foundation.
5.3 couples of hSODlG93ANSC34 cell has protective effect
To inquire into the influence of Quzhazhigan, piceatannol, resveratrol to ALS cell model, in hSODlG93AWith
hSODlWTQuzhazhigan, piceatannol, resveratrol on NSC34 cell using various concentration carry out intervention processing, and observation is thin
Born of the same parents' form and MTT vigour changes.10,20,40 μm of olL are given into the cell in the NSC34 of transfection-1Quzhazhigan, white skin China fir
After alcohol, resveratrol culture 48h, discovery cellular morphology is without significant change, but 20,40 μm of olL-1Quzhazhigan, white skin
Cell number after China fir alcohol intervention increased (P < 0.05) compared with the cell number of non-administered group；Resveratrol has increase trend,
But no difference of science of statistics is shown in Fig. 2.
It is horizontal that 5.4 Quzhazhigans reduce MDA
It is in view of Quzhazhigan, piceatannol still unclear to the mechanism of action of ALS, the normal culture of this measuring
NSC34 cell and hSODlG93AThe water of the malondialdehyde (Malonaldehyde, MDA) of NSC34 cell each group
Flat variation.Experimental result finds the hSODl compared with the NSC34 cell normally cultivatedG93AMDA content obviously rises in NSC34 cell
High (P < 0.001), and giving 20,40 μm of olL-1Quzhazhigan, piceatannol administration after, MDA content be substantially reduced (P <
0.01), and certain dosage correlation is presented；And resveratrol is shown in Fig. 3 to the reduction no difference of science of statistics of MDA content.
2 Antioxidation in vitro of embodiment
1 test sample
Quzhazhigan, molecular weight 406, white crystals or crystalline powder, purity 99.6%, lot number 20120402；
Piceatannol, molecular weight 244, red-brown powder, purity 99.8%, lot number 906099a；
Resveratrol, molecular weight 228, white powder, purity 99.8%, lot number CY120325.
The above sample is provided by drug research institute of Kun Yao group, and room temperature sealing is kept in dark place.When test, with 1/,100,000
Electronic analytical balance weighs, and with DMSO dissolution, prepares and dilutes, and controls the concentration of DMSO in each detection architecture 1%.
2 reference substances
Vitamin C, purity 99.5%, CSPC Weisheng Pharmaceutical (Shijiazhuang) Co., Ltd., lot number 1130172056；
Edaravone Injection, 30mg/20mL, Kunming Jida Pharmaceutical Co., Ltd., lot number 131202.
3 reagents
Pyrogallic acid (pyrogallol), Chinese medicines group Solution on Chemical Reagents in Shanghai Co., Ltd (lot number: 20140320)；Pheno
Piperazine Methylsulfate (PMS), Beijing DingGuo ChangSheng Biology Technology Co., Ltd, Genview packing, Lot:3AE10130；Also
Prototype cozymase (NADH), 1g, BIOSHARP, Exp:2017/11；Nitro tetrazolium blue (NBT), 100mg, BIOSHARP, Exp:
2016/11；Hexichol is for bitter taste hydrazine free radical (DPPH), Lot:WA0808EA14, Shanghai Yuan Ye Biotechnology Co., Ltd；
ABTS, Lot:SLBH2992V, Sigma；Phenol, Lot:LJ0607S10010J, Sangon Biotech (Shanghai) Co., Ltd.；4-
Amino-antipyrine (4-AA), Lot:YK0709B1013J, Sangon Biotech (Shanghai) Co., Ltd.；Horseradish peroxidase
(POD), Lot:LJ0731YA14, Activity >=300U/mg, Shanghai Yuan Ye Biotechnology Co., Ltd.Dense HCL
(20090420), NaOH (20091001), KOH (20110223), the Yunnan Industry Development Area Yang Lin Shan Dian pharmaceutcal corporation, Ltd；
KH2PO4(20090304), Shantou City reaches Hao fine chemicals Co., Ltd；FeSO4·7H2O (20110223), Chinese medicines group
Learn reagent Co., Ltd；Na2HPO4·12H2O (20131120) Tianjin Rui Jin spy Chemical Company, AR；Trichloroacetic acid
(TCA) (20131105), Guangdong Guanghua Science and Technology Co., Ltd.；K2PO4.3H2O (0100722), the potassium ferricyanide [K3Fe
(CN)6](20140510)、FeCl3·6H2O(20131020)、NaH2PO4·2H2O (20120202), potassium peroxydisulfate (K2S2O8)、
30%H2O2(20140210), Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd..Complete pH calibrates buffer, and Shanghai City love is built
Chemical reagent work, lot number 0920515；Tris-base, Purity >=99.9%, Amresco, Exp2015/12；DMSO, GC99.5%,
083K0135, Sigma.
4 instruments and consumptive material
PowerWave XS2 all-wave length microplate reader, BioTek；UB-7 type pH meter, Denver's instrument (Beijing) Co., Ltd；
Elix water purification machine, Milii-Q ultrapure water machine, Millipore；AC211S electronic analytical balance, Sartorius；DHG—9245A
Type electric drying oven with forced convection, Shanghai Yiheng Scientific Instruments Co., Ltd；SK-8200H ultrasonic cleaner, Shanghai section lead Ultrasound Instrument
Device Co., Ltd；0.5~10 μ L, 10~100 μ L, 200~1000 μ L single track micropipettors, Eppendorf；50~300 μ L
8 micropipettors, BioScience；0.2~10 μ L, 5~120 μ L single track electric pipettors and 10~300 μ L 8 are electronic
Pipettor, Biohit；Other glass apparatus and consumptive material, it is domestic.
5 test methods and result
5.1 couples of O2 ?The influence of Scavenging activity
This test uses PMS-NADH-NBT system[10-11], each reaction reagent is sequentially added by table 1, mixes postposition room temperature
It is protected from light 5min, is returned to zero with blank well, OD value is measured at 560nm, every concentration is arranged 3 holes and repeats, and as a result takes mean value, calculates
O2 ?Clearance rate and IC50.It the results are shown in Table 2.
1 PMS-NADH-NBT reaction system of table
| Reaction reagent | Blank well (μ L) | Gauge orifice (μ L) | It measures hole (μ L) |
| Test liquid | ? | ? | 50 |
| Solvent (10%DMSO) | 50 | 50 | ? |
| 50mMTris-HCl, pH8.0 | 160 | 160 | 160 |
| 1mM NADH | 20 | 20 | 20 |
| 0.5mM NBT | 20 | 20 | 20 |
| 10μM PMS | ? | 50 | 50 |
| H2O | 50 | ? | ? |
2 series of samples of table is to O2 ?Scavenging activity
| Title | IC50(μM) |
| Piceatannol | 5.81 |
| Quzhazhigan | 419.00 |
| Resveratrol | 250 |
| Vc | / |
| Edaravone | 13.85 |
As can be seen from Table 2, piceatannol and Edaravone are to O2 ?Scavenging activity it is close, IC50Respectively 5.81,13.85
μM；Quzhazhigan, resveratrol have certain scavenging effect, and Vc fails to measure IC50。
The influence of 5.2 pairs of OH Scavenging activities
This experimental evidence POD and H2O2The principle that OH is generated in reaction process, using POD-AA-Phenol colour developing body of laws
System[12-13], each reaction reagent is sequentially added by table 3,37 DEG C of reaction 10min, are returned to zero after mixing with blank well, are surveyed at 505nm
Determine OD value, as a result every 3 hole of concentration mensuration takes mean value.It the results are shown in Table 4.
Table 3POD-AA-Phenol development process reaction system
| Reaction reagent | Blank well (μ L) | Gauge orifice (μ L) | It measures hole (μ L) |
| Test sample | ? | ? | 50 |
| Solvent (10%DMSO) | 50 | 50 | ? |
| 50mMTris-HCL, pH7.4 | 155 | 155 | 155 |
| 2%4-AA | 15 | 15 | 15 |
| 0.94%Phenol | 15 | 15 | 15 |
| 0.1U/mLPOD | 15 | 15 | 15 |
| 2mM H2O2 | ? | 50 | 50 |
| H2O | 50 | ? | ? |
Scavenging activity of 4 series of samples of table to OH
| Title | IC50(μM) |
| Piceatannol | 9.29 |
| Quzhazhigan | 36.82 |
| Resveratrol | 50.24 |
| Vc | 27.17 |
By table 4 as it can be seen that piceatannol is most strong to OH Scavenging activity, IC50It is 9.29 μM；Vc, Quzhazhigan take second place, white Chenopodiaceae
Reed alcohol is most weak, IC50Respectively 27.2 μM, 36.82 μM, 50.24 μM.
5.3 couples of ABTS.+The influence of Scavenging activity
Reference literature uses TEAC method, sequentially adds each reaction reagent by table 5, and room temperature is protected from light 10min after mixing, with
Blank well zeroing, measures OD value, as a result every 3 hole of concentration mensuration takes mean value at 734nm.It the results are shown in Table 6.
Table 5ABTS reaction system
| Reaction reagent | Blank well (μ L) | Gauge orifice (μ L) | It measures hole (μ L) |
| Test sample | ? | ? | 50 |
| Solvent (10%DMSO) | 50 | 50 | ? |
| ABTS reaction solution | ? | 250 | 250 |
| 95%ETOH | 250 | ? | ? |
6 series of samples of table is to ABTS.+Scavenging activity
| Title | IC50(μM) |
| Piceatannol | 11.12 |
| Quzhazhigan | 4.28 |
| Resveratrol | 4.47 |
| Vc | 15.52 |
By table 6 as it can be seen that Quzhazhigan is to ABTS.+With stronger Scavenging activity, IC50It is 4.28 μM；Resveratrol, white skin
China fir alcohol takes second place with Vc, IC50Respectively 4.47,11.12 and 15.52 μM.
The influence of 5.4 pairs of total reducing powers
This test uses FeCl3Reduction method is sequentially added each reaction reagent by table 7, is returned to zero with blank well, at 700nm
OD value is measured, as a result every 3 hole of concentration mensuration takes mean value.Separately take the FeSO of 0.06,0.12 ... 3.7mM concentration430 μ L of solution production
Standard curve (is shown in Table 8 and Fig. 4), will make 1 μM of Fe in reaction system3+It is reduced to Fe2+Ability be defined as 1 also original unit,
Calculating each tested material makes 168.2 μM of Fe in reaction system3+Half is reduced to Fe2+EC50.It the results are shown in Table 9.
7 TOC of table measures reaction system
8 various concentration FeSO of table4Product and absorbance value in the reaction system
Influence of 9 series of samples of table to total reducing power (TOC)
| Title | EC50(μM) |
| Piceatannol | 38.22 |
| Quzhazhigan | 127.10 |
| Resveratrol | 136.09 |
| Vc | 80.18 |
| Edaravone | 94.55 |
By table 9 as it can be seen that piceatannol reducing power is most strong, EC50It is 38.22 μM；Quzhazhigan and Vc, Edaravone are made
With suitable, resveratrol is most weak, EC50Respectively 127.1,80.18,94.55,136.09 μM,.
To sum up, Quzhazhigan is most strong to the scavenging effect of ABTS+ in vitro, and piceatannol takes second place；Removing O2 ?·、
OH and the aspect piceatannol effect of total reducing power are most strong, and Quzhazhigan takes second place.
Effect of the embodiment 3 to glutamate induction neural information encoding
1 experimental material
1.1 given the test agent
Quzhazhigan, molecular weight 406, white crystals or crystalline powder, purity 99.6%, lot number 20120402；
Piceatannol, molecular weight 244, red-brown powder, purity 99.8%, lot number 906099a；
Resveratrol, molecular weight 228, white powder, purity 99.8%, lot number CY120325.
Preparation method: tested material is first configured to 10mM mother liquor with DMSO, then with normal saline dilution to required concentration.
1.2 negative controls
Sodium chloride injection (0.9% physiological saline), Qingzhou Yaowang Pharmaceutical Co., Ltd., lot number: 2215110901, rule
Lattice: 500ml/ bottles.
1.3 positive reference substance
Edaravone Injection, Nanjing Xianshengdongyuan Pharmaceutical Co., Ltd, lot number: 80-170102, specification: 5ml:10mg.
1.4 SH-SY5Y human neuroblastoma cells
Human brain neuroblastoma, metastasis site marrow, growth characteristics: adherent growth, it is limited that auspicious biotechnology is answered in Shanghai
Company.
1.5 laboratory apparatus
Full-automatic microplate reader, the production of Labsystem Dragon company；CO2Incubator, German Heaeus company production；?
Set phase contrast microscope, the production of Metrio company.
1.6 other reagents
Tetrazole indigo plant (MTT): the production of Sigma company, lot number: 67R338V；DMEM culture solution: the production of BI company, lot number:
0035316；D-Hanks buffer: the production of BI company, lot number: 0022016；Fetal calf serum: PAN Biotech production, lot number:
P140107；Blueness-streptomysin: Hyclone production, lot number: J140055；Pancreatin: Corining production, lot number: 12616003；Paddy
Propylhomoserin: the production of Sigma company, lot number: BCBG7324V.
2 experimental methods and result
Cell is divided into normal culture group, model group, administration group and positive controls.Cell is after drug-treated, except normal
Outside group and model group, after each administration group first gives various concentration tested material pretreatment 0.5h, then for 24 hours with the incubation of 100mM glutamic acid.
Make MTT analysis, afterwards for 24 hours to measure cell survival rate.Specifically, 5mg/ml MTT solution is added in every hole, keeps its final concentration of
0.5mg/ml, continues to cultivate 4h in incubator, then abandons training liquid, and every hole is added 200 μ l DMSO, light is read in microplate reader
Density OD value (measurement wavelength 570nm).According to formula calculated result:
All data are indicated with means standard deviation.Statistics compares to be analyzed using ANOVA, and group difference uses Fisher
LSD, which is examined, to carry out.
The results are shown in Table 10, and glutamic acid can dose-dependent damaged nerve cells.100mM dosage is selected in conjunction with document
Exitotoxicity damage dose as follow-up study.
As shown in table 11, glutamic acid causes the exitotoxicity of nerve cell to damage under 100mM dosage, and this effect can be
Positive drug Edaravone (100 μM) is fought.Quzhazhigan and piceatannol have significant confrontation Nervous toxicity under 300 μM of dosage
Property effect；Resveratrol effect is unobvious.
Exitotoxicity of 10 glutamic acid of table to SY5Y nerve cell
**P < 0.01vs negative control
Effect of the different tested materials of table 11. to glutamate induction SY5Y nerve cell exitotoxicity
**, p < 0.01vs negative control group；##, p < 0.01vs model group
3 conclusions
The result shows that Quzhazhigan and piceatannol are 300 for the SY5Y cellular neural exitotoxicity of glutamate induction
Neurotoxic effect can be significantly fought under μM dosage, resveratrol effect is unobvious.
Embodiment 4, on Level In Rats With Focal Cerebral Ischemia Neuron Apoptosis and regenerated influence
1 experimental material
1.1 given the test agent
Injection Quzhazhigan, Kun Yao Group Plc, specification: 20mg/ branch (every 20mg containing Quzhazhigan,
Auxiliary material hydroxy propyl-Beta cyclodextrin 100mg), lot number: 20121031-01
It prepares: taking one, be dissolved to 2mL with 0.9% sodium chloride injection, obtain 10mg/mL solution, and dilute to obtain 2mg/mL
Solution, it is spare.
1.2 reagent
TUNEL kit, Roche, lot number 10348000；BrdU:Sigma, lot number HMBD2252V；
Anti- BrdU antibody: BioRAD, lot number 0413；Anti- rat Ki-67570 antibody: eBioscience, batch
Number E16297-102；Anti- Bcl-2 antibody: Cell Signaling Technology, lot number 16；Anti-bax antibodies: Cell
Signaling Technology, lot number 14；Anti- Cleaved Caspase-3 antibody: Cell Signaling
Technology, lot number 19；Alexa Fluor568 donkey anti-rabbit IgG (H+L): Molecular Probes, lot number 1134929；
Alexa Fluor568 goat anti rat IgG (H+L): Molecular Probes, lot number 1259374；Iba-1:Wako, lot number
019-19741；Triton X-100:Sigma, lot number 031M0301V；β-Actin (C4)-HRP:Santa Cruz
Technology, lot number F2614；Goat anti-Rabbit IgG-HRP:Santa Cruz Technology, lot number
J2414；Goat anti-Mouse IgG-HRP:Santa Cruz Technology, lot number G3114；BCA protein quantification reagent
Box: Shanghai Wei Ao Biotechnology Co., Ltd, lot number WB0123；Trizma base:Sigma, lot number WXBB23800；
Glycerol: raw work biology, lot number 0830SJ80；DTT: Shanghai Wei Ao Biotechnology Co., Ltd, lot number WB0145；Acryloyl
Amine: raw work biology, lot number 10798524；Bis-Acralamide: raw work biology, lot number 10796524；SDS:Amresco, batch
Number 0227；Glycine: Amresco, lot number 0176；APS: Shanghai Wei Ao Biotechnology Co., Ltd, lot number WB0142；TEMED:
Shanghai Wei Ao Biotechnology Co., Ltd, lot number WB0150；Tween-20: raw work biology, lot number 0710SJ100；Pvdf membrane:
Millipore, lot number K2PA0943DK；BSA: Shanghai Wei Ao Biotechnology Co., Ltd, lot number WH1044；The super quick colour developing of ECL
Liquid: Shanghai Wei Ao Biotechnology Co., Ltd, lot number WB0164；
1.3 experimental animal
SD rat, male, weight: 230~250 grams；Shanghai Slac Experimental Animal Co., Ltd., animal productiong licensing
Number: SCXK (Shanghai) 2012-0002.
Raising: animal feeding is in positive pressure purified and ventilated animal house, and 24 ± 1 DEG C of room temperature, humidity 40~70%, artificial light
Simulate day-night change, feed food and water freely.
1.4 laboratory apparatus
Western electrophoresis power: BG-Power 600K, Beijing Baijing Biotechnology Co., Ltd.；FluorChem E at
As system: Protein Simple, Santa Clara, CA, USA；Fluorescence microscope: SMZ800, Nikon.2 experimental methods and
As a result
2.1 experimental group
Healthy male SD rat 60 is only divided into 3 groups, every group 20, respectively sham-operation group (0.9% sodium chloride injection),
Model group (0.9% sodium chloride injection), Quzhazhigan group (4mg/kg).
Administration route and administration time: each group is injected intravenously administration, administered volume 2mL/kg when Reperfu- sion.
2.2 model copy
2.2.1 nerve cell apoptosis is tested
By rat anesthesia, all blood flow sources of surgical interruption right side MCA.Standby line is tightened, after 2 hours, sublingual vein is given
Nylon wire is extracted after giving each group drug, leads to its blood flow again, standby line and skin suture is ligatured, operated rats is put back in cage and are raised
It supports.After filling again for 24 hours, after (every group 10) 12% chloral hydrate anesthesias of intraperitoneal injection of Some Animals, with 0.9% sodium chloride injection
Through aorta ascendens systemic perfusion, atrium dextrum clip, until efflux is colourless, then fill with 4% paraformaldehyde；It is taken after perfusion fixation big
Brain, and the rear fixed 12-16h in same fixer.The brain fixed carries out frozen section, is placed in and freezes in protection liquid, and -20
It DEG C saves backup, to carry out Cleaved Caspase-3 and Ki-67 immunohistochemical staining, TUNEL dyeing.Other part
Animal (every group 10) directly takes brain, and -80 DEG C save backup, to carry out Bax and Bcl immunoblot experiment.
2.2.2 nerve cell regeneration tests
Acute stage experiment: rat by 2.2.1 method carry out model copy, given while ischemic BrdU (10mg/kg,
1mL/kg), then after filling for 24 hours, animal takes brain row frozen section through paraformaldehyde perfusion fixation, to carry out BrdU immunohistochemistry
Dyeing.
Chronic experiment: rat by 2.2.1 method carry out model copy, given while ischemic BrdU (10mg/kg,
1mL/kg), once a day, totally 14 days.After last time gives BrdU for 24 hours, perfusion fixation is carried out, brain row frozen section is taken,
To carry out BrdU immunohistochemical staining.
2.3 evaluation index
2.3.1 to the influence of Neuron Apoptosis
Apoptotic cell (TUNE method) and signaling molecule (such as Caspase-3 relevant to apoptosis are had detected in our current research
With Bax etc.).
Shown in table 12: compared with sham-operation group, model group rats cerebral ischemia-fill again after for 24 hours, intracerebral TUNEL method dyeing mark
The apoptotic cell and Cleaved Caspase-3 immuno positive (Cleaved Caspase-3-ir) cell of note increase
(P < 0.01), Quzhazhigan 4mg/kg group can significantly reduce intracerebral TUNEL method dye marker apoptotic cell and
Cleaved Caspase-3 immuno positive (Cleaved Caspase-3-ir) cell (compared with model group, P < 0.05 or P <
0.01)。
Influence of the table 12. to apoptosis amount
*P<0.05,**P < 0.01vs sham-operation+NS；#P<0.05,##P < 0.01vs model+NS
2.3.2 to the influence of neuronal survival
It is as shown in table 13: compared with sham-operation group, model group animal brain transient ischemia/reperfusion for 24 hours after, the level of intracerebral Ki67 is aobvious
Work increases (P < 0.01), the trend that Ki67 is reduced after Quzhazhigan 4mg/kg administration, but there was no significant difference (with model group ratio
Compared with P > 0.05).While transient ischemia/reperfusion, BrdU is injected intraperitoneally, then after filling for 24 hours, brain area can observe in ischemic side
BrdU level significantly increase (P < 0.01vs sham-operation group), this effect can be given by vein Quzhazhigan 4mg/kg pair
Anti- (P < 0.01vs model group).It is as shown in table 14: to give within continuous two weeks BrdU, the level of model group animal ischemic side brain area BrdU
Then substantially reduce, and the level for giving Quzhazhigan group rat ischemia side brain area BrdU then increased significantly (P < 0.05vs model
Group).The result shows Quzhazhigan has the function of promoting cell Proliferation.
Influence of the table 13 to brain cell proliferation
| Group | Dosage (mg/kg) | N | Ki67 (acute) | BrdU (acute) |
| Sham-operation+NS | -- | 4 | 29.2±20.1 | 15.6±14.5 |
| Model+NS | -- | 6 | 59.2±30.2** | 43.2±24.8** |
| Quzhazhigan | 4 | 6 | 43.1±17.1 | 24.7±17.7## |
*P<0.05,**P < 0.01vs sham-operation+NS；#P<0.05,##P < 0.01vs model+NS
The influence of brain cell proliferation after table 14 fills rat ischemia 2 weeks again
| Group | Dosage (mg/kg) | N | BrdU (chronic) |
| Sham-operation+NS | -- | 5 | 59.4±57.7 |
| Model+NS | -- | 7 | 16.3±15.4* |
| Quzhazhigan | 4 | 5 | 53.2±49.7# |
*P < 0.05,**P < 0.01vs sham-operation+NS；#P < 0.05,##P < 0.01vs model+NS
2.4 brief summary
Quzhazhigan 4mg/kg can significantly reduce TUNEL method dyeing mark in line brush induction rat focal cerebral ischemia
The apoptotic cell and Cleaved Caspase-3 immuno positive (Cleaved Caspase-3-ir) cell of note prompt bent letter
Stilbene glycosides 4mg/kg has the function of anti-Neuron Apoptosis；Chronic cerebral ischemia in rats ischemic side brain area BrdU can be made horizontal obvious simultaneously
Increase, prompts Quzhazhigan that there is obvious repair to nerve cell.
To sum up, Quzhazhigan and its derivative, which have, inhibits toxicity of excitatory amino acid, anti-oxidant, anti-nerve cell to wither
It dies, promote the neuroprotections such as Neuronal Survival, proliferation, according to the possible pathogenesis of ALS, Quzhazhigan and its derivative
Object can be intervened for the above pathogenesis, play preventive and therapeutic action to ALS.With good potential applicability in clinical practice.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. application of the Quzhazhigan or derivatives thereof in the preparation of preparation protection nerve cell.
2. application according to claim 1, which is characterized in that the protection is horizontal to reduce MDA in nerve cell.
3. application according to claim 1, which is characterized in that the protection is raising nerve cell vigor.
4. described in any item applications according to claim 1~3, which is characterized in that the nerve cell is neuronal cell；Institute
Stating neuronal cell is normal neuronal cell, or transfection hSODlG93ANeuronal cell.
5. application according to claim 1, which is characterized in that the protection is neural to inhibit neural information encoding, inhibition
The apoptosis of cell and/or the proliferation for promoting nerve cell.
6. described in any item applications according to claim 1~5, which is characterized in that the derivative of the Quzhazhigan is white skin
China fir alcohol.
7. Quzhazhigan or derivatives thereof is applied in the drug of preparation prevention and treatment motor neuron disease.
8. application according to claim 7, which is characterized in that the motor neuron disease is amyotrophic lateral sclerosis.
9. according to the described in any item applications of claim 7~8, which is characterized in that the derivative of the Quzhazhigan is white skin
China fir alcohol.
10. a kind of drug for preventing and treating motor neuron disease, which is characterized in that including at least one in Quzhazhigan or derivatives thereof
Kind.
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| WO2021051271A1 (en) * | 2019-09-17 | 2021-03-25 | 深圳大学 | Use of piceatannol in preparing a drug for preventing and treating neurodegenerative diseases |
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