CN104922096A - Application of 4-methoxy benzyl alcohol to preparation of antioxidant type drug - Google Patents
Application of 4-methoxy benzyl alcohol to preparation of antioxidant type drug Download PDFInfo
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- CN104922096A CN104922096A CN201510278051.5A CN201510278051A CN104922096A CN 104922096 A CN104922096 A CN 104922096A CN 201510278051 A CN201510278051 A CN 201510278051A CN 104922096 A CN104922096 A CN 104922096A
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Abstract
The invention discloses an application of 4-methoxy benzyl alcohol to preparation of an antioxidant type drug. By reducing an in-cell ROS (reactive oxygen species) level, improving SOD (superoxide dismutase) activity, increasing GSH (glutathione) content and/or resisting cell apoptosis, the 4-methoxy benzyl alcohol can play a role of resisting oxidative damage and thus improves cell viability. The 4-methoxy benzyl alcohol can be prepared into an antioxidant for preventing oxidative damage and is good in application prospect.
Description
Technical field
The present invention relates to 4-methoxyl group benzylalcohol and prepare the purposes in antioxidant medicine.
Background technology
Oxidative stress (Oxidative Stress) refer to vivo oxidation and antioxidation unbalance, tend to oxidation, cause neutrophilic granulocyte inflammatory infiltration, protease secretion increase, produce a large amount of intermediate oxidation product.Oxidative stress is the potential injury that the balance destroying strong oxidizer and antioxidant causes, and the destruction of oxidant, antioxidant balance is the main cause of cell injury.Oxidative stress is a kind of negative effect produced in vivo by free radical, and is considered to the key factor causing senescence and disease.In addition, cerebral ischemia reperfusion injury (cerebral ischemia reperfusion injury, CIRI) be ischemic cerebrovascular (Ischemic cerebrovascular diseases, ICVD) main pathological process, and oxidative stress (oxidative stress, OS) be the key factor causing CIRI, therefore, antioxidant stress injury is also significant to the Treatment and Prognosis of ICVD.
4-methoxyl group benzylalcohol, colourless or micro-yellow liquid.Fusing point 23-25.5 DEG C, boiling point 159 DEG C, relative density 1.113 (15/15 DEG C), index of refraction 1.5442.Be soluble in alcohol and ether, water-soluble hardly, its chemical formula is: C
8h
10o
2, structural formula is
current 4-methoxyl group benzylalcohol eats usually used as flavorant, has no it and reports for antioxidative.
Summary of the invention
4-methoxyl group benzylalcohol is the object of the present invention is to provide to prepare the purposes in antioxidant.
Antioxidant (AOA) be also scavenger, is startup and hypertrophy (spreading) process that a class can suppress or block free chain reaction, reduces number of free radical, delays the old process of sorrow, improve the compound of vitality.
The present invention provide firstly 4-methoxyl group benzylalcohol and is preparing the purposes in antioxidant medicine.
Further, described antioxidant medicine is the medicine of anti-oxidative damage.
Further, the medicine of described anti-oxidative damage is the medicine preventing and/or treating oxidativestress damage.
Again further, described medicine is the medicine reducing ROS level, improve SOD vigor, improve GSH content and/or anti-apoptotic.
ROS: active oxygen; SOD: superoxide dismutase activity; GSH: glutathion.
Wherein, described preparation is oral formulations.
Wherein, described oral formulations is capsule, tablet.
Present invention also offers a kind of medicine of anti-oxidative damage, it be with 4-methoxyl group benzylalcohol for active component, add the preparation that pharmaceutically acceptable adjuvant is prepared from.
Described preparation is oral formulations.
4-methoxyl group benzylalcohol is by the effect reducing intracellular ROS level, improve SOD vigor, improve GSH content and/or anti-apoptotic performance anti-oxidative damage, thus raising cell survival rate, can prepare and become antioxidant, for preventing anti-oxidative damage, application prospect is good.
Below by detailed description of the invention, the present invention is described in further details, but be not limitation of the present invention, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
Accompanying drawing explanation
Fig. 1 4-methoxyl group benzylalcohol is to H
2o
2mMP change (40 × 10) after damage PC12 cell.
Fig. 2 4-methoxyl group benzylalcohol is to H
2o
2after causing PC12 cell oxidative damage.The impact (note: 1Control, 2Model, 3Eda-160,4MA-20,5MA-40,6MA-80) that Caspase-9, Caspase-12, Caspase-3 express.
Fig. 3 4-methoxyl group benzylalcohol is to H
2o
2cause the impact of ERK1/2, P-ERK1/2, JNK, P-JNK, p38, P-p38 protein expression after PC12 cell oxidative damage.(note: 1Control, 2Model, 3Eda-160,4MA-20,5MA-40,6MA-80,7Model+ inhibitor, 8Eda-160+ inhibitor, 9MA-20+ inhibitor, 10MA-40+ inhibitor, 11MA-80+ inhibitor).
Detailed description of the invention
The antioxidation of experimental example 1 4-methoxyl group benzylalcohol
The effect of Part I, anti-oxidative damage
1 experiment material
1.1 experimental cell
Differentiated PC12 cells strain (PC12 cell), purchased from Shanghai Inst. of Life Science, CAS.
1.2 experimental drug
1.3 experiment reagent
1.4 experimental apparatus
1.5 main agents preparations
1.5.1 complete medium
FBS and modified form RPMI-1640 culture medium, with the ratio of 1:9, are formulated as the RPMI-1640 complete medium containing 10%FBS, add 100U/mL Pen .-Strep solution simultaneously, seal 4 DEG C and save backup.
1.5.2PBS buffer
Get 9.88g PBS liquid mixed powder and be dissolved in 1L distilled water, adjust PH to 7.4 after dissolving completely, filtration sterilization, 4 DEG C save backup.
1.5.3MTT solution
MTT dry powder 0.25g, adds 50mL PBS buffer, filtration sterilization after dissolving completely, 2mL subpackage, and-20 DEG C save backup, omnidistance lucifuge operation.
1.5.4H
2o
2modeling liquid
Get 30%H
2o
2stock solution 1mL adds 9mL aseptic double-distilled water, mixes gently, gets the H after mixing
2o
2liquid 0.1mL adds 9.9mL aseptic double-distilled water, mixes gently, then gets the rear H of second time mixing
2o
2liquid 1mL adds 9mL aseptic double-distilled water, mixes gently and obtains H
2o
2the high concentration storing solution (1mmol/L) of modeling liquid, process for preparation and each concentration H after preparing
2o
2solution all notes strict lucifuge and sterile working, and posts 4 DEG C, label and keep in Dark Place, and gets H before use
2o
2the high concentration storing solution of modeling liquid joins in the serum-free RPMI-1640 culture medium of 37 DEG C of warm mistakes mix gently repeatedly in the ratio of 1: 19, obtains the H that concentration is 50 μm of ol/L
2o
2modeling liquid, H
2o
2modeling liquid is now with the current, how much need join how many.
1.6 Pharmaceutical formulations
1.6.1 4-methoxyl group benzylalcohol
Precision takes appropriate 4-methoxyl group benzylalcohol and adds DMSO and be mixed with 8.0 × 10
4the high concentration storing solution of μ g/mL, often pipe 30 μ L quantitative separating,-20 DEG C keep in Dark Place, the maximum concentration that an effective serum-free RPMI-1640 culture medium dilution is mixed with required administration concentration group is got before experiment, filtration sterilization, be diluted to all the other required administration group concentration successively by this concentration, must ensure that the DMSO final concentration of each group is consistent, and final concentration be less than 0.2%.
1.6.2 Edaravone (Edaravone Injection)
Edaravone is a kind of cerebral protective agent (free radical scavenger), anti-lipid peroxidation, thus suppresses the oxidative damage of brain cell, vascular endothelial cell, neurocyte.
Precision takes Edaravone 32mg and joins in 20mL serum-free RPMI-1640 culture medium, whole dissolving is stirred to gently in the water-bath of 55 DEG C, filtration sterilization in sterile biological safety cabinet, obtain the Edaravone storing solution that concentration is 1.6mg/mL, 4 DEG C keep in Dark Place, press desired concn conversion dilution ratio before use, carry out dilution administration by serum-free RPMI-1640 culture medium.
2 experimental techniques
The recovery of 2.1PC12 cell, Secondary Culture and frozen
2.1.1PC12 the recovery of cell
From-80 DEG C of refrigerators, take out a PC12 cell cryopreservation tube put into the jolting fast of 37 DEG C of water immediately, until melt completely, spray 75% alcohol disinfecting outer wall, cell suspension is moved into the 25cm that the complete RPMI-1640 culture medium of 5mL is housed in Biohazard Safety Equipment
2in culture bottle, shake up gently and be placed on 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2cultivate in incubator, change liquid after 4h, after spending the night, change liquid again.
2.1.2PC12 the Secondary Culture of cell
When the PC12 cell cultivated wait recovering grows to the adherent rate of 70%-80%, abandon former culture medium, get the cleaning of 2mL PBS liquid once, add 0.25% pancreatin 2mL to be placed in incubator and to digest 3min, wave and culture bottle makes cell detachment bottle wall, basis of microscopic observation is when cellular contraction change circle, after departing from bottle wall, add the complete RPMI-1640 culture medium of 2mL and stop digestion, with the centrifugal 5min of 1000r/min, abandon supernatant liquid, add the complete RPMI-1640 culture medium of 2mL, even piping and druming cell dispersion makes cell suspension, sub-bottle goes down to posterity, in Secondary Culture process, about 2d changes a fresh medium, take the logarithm trophophase cell for experiment.
2.1.3PC12 cell is frozen
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in 2.1.2 is 10
6the cell suspension of level, the DMSO of 0.9mL suspension and 0.1mL 100% is added in the cryopreservation tube of each 1.5mL, the cryopreservation tube of good seal evenly, is put into program temperature reduction box and is directly put into-80 DEG C of refrigerators by piping and druming, moves in-80 DEG C of refrigerators and preserve in common freezing storing box after spending the night.
2.2 4-methoxyl group benzylalcohols are on the impact of oxidative damage PC12 cell survival rate
2.2.1 method
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in 2.1.2 is 3 × 10
4the cell suspension of individual/mL, is inoculated on 96 orifice plates, is placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2after cultivating 24h in incubator, be divided into Normal group, H
2o
2damage, Edaravone group (160 μ g/mL) and 4-methoxyl group benzylalcohol (20 μ g/mL, 40 μ g/mL, 80 μ g/mL) three dosage groups, often group establishes 6 multiple holes, respectively after administration 24h, except Normal group, all the other each group H all adding 50 μm of ol/L
2o
2modeling liquid, damage 2h, then careful suction abandons original fluid, slowly adds containing serum RPMI-1640 culture fluid, be placed in CO along hole wall edge
2continue in incubator to cultivate 42h, measure cell survival rate with mtt assay.
2.2.2 result
Compare with normal group, model group cell quantity reduces, and survival rate reduces (P<0.01); Compare with model group, the cell survival rate under Edaravone 160 μ g/mL and 4-methoxyl group benzylalcohol 20 μ g/mL, 40 μ g/mL, 80 μ g/mL concentration significantly raises (P<0.01), the results are shown in Table 1.
Table 1 4-methoxyl group benzylalcohol is to H
2o
2cause the impact of cell survival rate after PC12 cell oxidative damage
n=6)
Note: compare with Control group and have statistical significance * * P<0.01
Compare with Model group and have statistical significance
##p<0.01
2.3 4-methoxyl group benzylalcohols are on the impact of oxidative damage PC12 intracellular ROS level
2.3.1 method
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in 2.1.2 is 2 × 10
5the cell suspension of individual/mL, is inoculated on 6 orifice plates, is placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2after cultivating 24h in incubator, be divided into Normal group, H
2o
2damage, Edaravone group (160 μ g/mL) and 4-methoxyl group benzylalcohol (20 μ g/mL, 40 μ g/mL, 80 μ g/mL) three dosage groups, often group establishes 3 multiple holes, respectively after administration 24h, except Normal group, all the other each group H all adding 100 μm of ol/L
2o
2modeling liquid, damage 2h, cleans 1 time with RPMI-1640, adds 0.5mL PBS, use cell scraper collecting cell, and piping and druming is even, the power of fluorescence after stimulating with fluorescence microplate reader detection under 488nm excitation wavelength and 525nm emission wavelength.
2.3.2 result
Compare with normal group, model group fluorescence intensity significantly strengthens (P<0.01); Compare with model group, Edaravone 160 μ g/mL and 4-methoxyl group benzylalcohol 20 μ g/mL, 40 μ g/mL, 80 μ g/mL each administration group fluorescence intensity all significantly weaken (P<0.05 or P<0.01), the results are shown in Table 2.
Table 2 4-methoxyl group benzylalcohol is to H
2o
2cause ROS level in cell after PC12 cell oxidative damage impact (
n=6)
Note: compare with Control group and have statistical significance * * P<0.01
Compare with Model group and have statistical significance
##p<0.01
2.4 4-methoxyl group benzylalcohols are on the impact of SOD activity in oxidative damage PC12 cell
2.4.1 method
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in 2.1.2 is 2 × 10
5the cell suspension of individual/mL, is inoculated on 6 orifice plates, is placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2after cultivating 24h in incubator, be divided into Normal group, H
2o
2damage, Edaravone group (160 μ g/mL) and 4-methoxyl group benzylalcohol (20 μ g/mL, 40 μ g/mL, 80 μ g/mL) three dosage groups, often group establishes 3 multiple holes, respectively after administration 24h, except Normal group, all the other each group H all adding 100 μm of ol/L
2o
2modeling liquid, damage 2h, cleans 1 time with RPMI-1640, adds 1mL sterilized water, adopt 3 multigelation cell lysis, and basis of microscopic observation is without intact cell, and the SOD got in cell pyrolysis liquid mensuration solution is active.
2.4.2 result
Measurement result shows: compare with normal group, and the active pole of the intracellular T-SOD of model group is markedly inferior to normal group (P<0.01); Compare with model group, Edaravone 160 μ g/mL and 4-methoxyl group benzylalcohol 20 μ g/mL, the intracellular T-SOD activity of 40 μ g/mL administration group, significantly higher than model group (P<0.01), the results are shown in Table 3.
Table 3 4-methoxyl group benzylalcohol is to H
2o
2cause T-SOD activity in cell after PC12 cell oxidative damage impact (
n=6)
Note: compare with Control group and have statistical significance * * P<0.01
Compare with Model group and have statistical significance
##p<0.01,
#p<0.05
2.5 4-methoxyl group benzylalcohols are on the impact of GSH content in oxidative damage PC12 cell
2.5.1 method
The same 2.4.2 of experimental technique, gets the GSH content in cell pyrolysis liquid mensuration solution.
2.5.2 result
Measurement result shows: compare with normal group, and the intracellular GSH content of model group is significantly lower than normal group (P<0.01); Compare with model group, Edaravone 160 μ g/mL and the intracellular GSH content of 4-methoxyl group benzylalcohol each administration group are all significantly higher than model group (P<0.01 or P<0.05), the results are shown in Table 4.
Table 4 4-methoxyl group benzylalcohol is to H
2o
2cause GSH content in cell after PC12 cell oxidative damage impact (
n=6)
Note: compare with Control group and have statistical significance * * P<0.01
Compare with Model group and have statistical significance
##p<0.01,
#p<0.05
2.6 4-methoxyl group benzylalcohols are on the impact of Caspase-3 activity in oxidative damage PC12 cell
2.6.1 method
Cell culture processes same 2.4.2, H
2o
2after damage 2h, clean 1 time with RPMI-1640, collecting cell, add 100 μ L lysates, resuspended precipitation, ice bath cracking 30min, whirlpool concussion therebetween 3 ~ 4 times, 4 DEG C centrifugal (12000r/min, 10min), gets supernatant and detects Caspase-3 enzymatic activity.
2.6.2 result
Measurement result shows: compare with normal group, and the intracellular Caspase-3 of model group obviously activates and increases (P<0.01); Compare with model group, in Edaravone 160 μ g/mL and 4-methoxyl group benzylalcohol each administration group cell, Caspase-3 activation degree is all starkly lower than model group (P<0.01), the results are shown in Table 5.
Table 5 4-methoxyl group benzylalcohol is to H
2o
2cause Caspase-3 activity in cell after PC12 cell oxidative damage impact (
n=6)
Note: compare with Control group and have statistical significance * * P<0.01
Compare with Model group and have statistical significance
##p<0.01
2.7 4-methoxyl group benzylalcohols are on the impact of oxidative damage PC12 mitochondrial membrane potential in anoxic
2.7.1 method
Cell culture and medication same 2.4.2, H
2o
2after damage 2h, clean 2 times with PBS, add 500 μ L JC-1 working solutions, be placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2hatch 20min in incubator, 1 × Buffer cleans 1 time, takes the photograph sheet in fluorescence microscopy Microscopic observation.
Normal cell: be viewed as yellow green under same optical filter
Apoptotic cell: be viewed as green under same optical filter
2.7.2 result
Normal group cell emitting fluorescence mostly is yellow green, and model group yellow-green fluorescence weakens, and mostly is green fluorescence, and MMP declines.Compare with model group, the cell green fluorescence under Edaravone 160 μ g/mL and 4-methoxyl group benzylalcohol 20 μ g/mL, 40 μ g/mL, 80 μ g/mL concentration reduces, and yellow-green fluorescence increases, and MMP obviously raises, as Fig. 1.
Part II is on the impact of oxidative damage PC12 cell death related protein
1 experiment material
1.1 experimental cell
Same Part I.
1.2 experimental drug
Same Part I.
1.3 experiment reagent
1.4 experimental apparatus
1.5Western-blot related reagent
1.5.1 10% Ammonium persulfate. (AP)
Accurately take Ammonium persulfate. 0.1g, be dissolved in 1mL distilled water, 4 DEG C save backup.Holding time is one week.
1.5.2 5 × electrophoretic buffer
Take 15.1g Tris, 94g glycine, 5g SDS, in beaker, adds distilled water and is settled to 1000mL, room temperature preservation after dissolving.Front distilled water is used to be diluted to 1 × electrophoretic buffer.
1.5.3 10 × transferring film buffer
Take 30.3g Tris, 151.1g glycine, add distilled water and be settled to 1000mL, room temperature preservation after dissolving.Use front distilled water to dilute 10 times, and add methanol to 20%.
1.5.4 10 × TBS buffer
Take 24.2g Tris, 80gNaCl, add distilled water and be settled to 1000mL, after dissolving, adjust pH to 7.6, room temperature preservation with hydrochloric acid.Use front distilled water to dilute 10 times, and add 1mL tween 20, be mixed with 1 × TBST buffer.
1.5.5 4X sample-loading buffer (loading buffer)
Accurately take bromophenol blue 0.04g, SDS 0.8g, add the mixing of 4mL glycerol, then add 1.0mol/L TrisHCl (pH6.8) 2mL, be finally settled to 10mL with distilled water, 1mL subpackage, 4 DEG C of preservations.1X is diluted to during use.
1.5.6 confining liquid
Accurately take 5g defatted milk powder, be dissolved in 1X TBST buffer, be settled to 60mL with 1X TBST buffer after dissolving completely, 4 DEG C save backup.
1.5.7 dithiothreitol, DTT (DTT) (10X)
Accurately take DTT3.09g, dissolve with 20mL 0.01mol/L sodium acetate solution (pH5.2), be distributed into 1mL aliquot and be stored in-20 DEG C, during use, be diluted to 1X.
1.5.8 lysate
In the ratio of RIPA lysate: PMSF=99:1, preparation cell pyrolysis liquid, matching while using.
2 experimental techniques
2.1 prepared by albumen sample
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in Part I 2.1.2 is 2 × 10
5the cell suspension of individual/mL, is inoculated on 6 orifice plates, is placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2after cultivating 24h in incubator, be divided into Normal group, H
2o
2damage group, Edaravone group (160 μ g/mL) and 4-methoxyl group benzylalcohol (20 μ g/mL, 40 μ g/mL, 80 μ g/mL) three dosage groups, often group establishes 3 multiple holes, respectively after administration 24h, except Normal group, all the other each group H all adding 100 μm of ol/L
2o
2modeling liquid, damage 2h, cleans 1 time with PBS, every hole adds 60 μ L lysates, ice bath cracking 5min, collecting cell lysate, 4 DEG C of centrifugal (12000r/min, 5min), get supernatant, separate 5 μ L for protein quantification, 10min is boiled in all the other sealings, every 13 μ L protein samples add 4 × sample-loading buffer 5 μ L and DTT 2 μ L and mix, 30 μ L subpackages ,-80 DEG C of Refrigerator stores.
2.2SDS-PAGE electrophoresis
2.2.1SDS-PAGE the preparation of glue
By description preparation SDS-PAGE separation gel and the concentrated glue of SDS-PAGE gel reagents box, as following table:
The preparation of table 6SDS-PAGE glue
2.2.2 encapsulating
Put into folder after glass plate alignment to clamp.Then be vertically stuck on gum-making rack, fixing safe, perfusion SDS-PAGE separation gel, during to proper height, seals with distilled water.After gelling to be separated is solid, discard water layer, perfusion SDS-PAGE concentrates glue, inserts comb.After gelling to be concentrated is solid, inserts in electrophoresis tank, add 1 × electrophoretic buffer, extract comb.
2.2.3 loading
Marker 5μL
Sample 10 μ about L
, there are 10 bands after Marker electrophoresis: 10,15,25,35,40,55,70,100,130,170Kda in the quality loadings such as each loading hole.
2.2.4 electrophoresis
Connect both positive and negative polarity by connection instruction, start electrophoresis, voltage is 150V, and during concentrated glue, electrophoretic current is 35mA, and during to sample to concentrated glue and separation gel demarcation line, electric current is adjusted to 80mA, and electrophoresis stops to bromophenol blue indicator when lowermost end.
2.2.5 transferring film
After stopping electrophoresis, according to the pre-dyed band cutting SDS-PAGE separation gel extremely suitable size of Marker, identical with glue size 2 filter paper and 1 pvdf membrane to be immersed in transferring film liquid about 10 minutes, pvdf membrane is inserted in absolute methanol before soaking and is activated 10min, assemble from down to up according to the order of filter paper-pvdf membrane-glue-filter paper, be placed in membrane-transferring device, 220mA transferring film 40min.
2.2.6 close
After transferring film terminates, take out pvdf membrane, be enclosed in confining liquid, room temperature shaker vibrations 1h.
2.2.7 antibody incubation
Primary antibodie confining liquid powder is diluted to debita spissitudo, is sealed in primary antibodie diluent by film, protein powder is placed on shaking table upward, 4 DEG C of overnight incubation, secondary daily 1X TBST at room temperature shaking table washes three times, each 5min.Film is sealed in two anti-diluents, protein powder is placed on shaking table upward, after incubated at room temperature 1h, wash three times with on 1X TBST at room temperature shaking table, each 5min.
Table 7 antibody dilution ratio
2.2.8 colour developing
ECL nitrite ion A and B mixed in equal amounts, be added on film, with MultiSpectral Image system Taking Pictures recording, with Photoshop and Image process band, obtains result.
2.8.9 date processing
The data SPSS19.0 software of experiment gained carries out statistical analysis, and result is with mean ± standard deviation
represent, adopt one factor analysis of variance (one-way ANOVE), variance is checked with LSD together, heterogeneity of variance Tamhane ' T
2inspection, with P < 0.05 for difference has statistical significance.
3 experimental results
Compare with normal group, model group Caspase-9, Caspase-12 and Caspase-3 express increases (P < 0.05 or P < 0.01), compare with model group, Edaravone 160 μ g/mL all has inhibitory action (P < 0.05) to the expression of Caspase-9, Caspase-12 and Caspase-3; The each administration group of 4-methoxyl group benzylalcohol also can suppress Caspase-9, Caspase-12 and Caspase-3, wherein, to the inhibition remarkable (P < 0.05 or P < 0.01) of Caspase-9 and Caspase-3, the results are shown in Figure 2, table 8.
Table 84-methoxyl group benzylalcohol is to H
2o
2cause Caspase-9, Caspase-12, Caspase-3 express after PC12 cell oxidative damage impact (
n=6)
Note: compare with Control group and have statistical significance * * P<0.01, * P<0.05
Compare with Model group and have statistical significance
##p<0.01,
#p<0.05
Part III 4-methoxyl group benzylalcohol is on the impact of oxidativestress damage PC12 cell MAPK signal path
1 experiment material
1.1 experimental cell
Same Part I.
1.2 experimental drug
Same Part I.
1.3 experiment reagent
1.4 experimental apparatus
Same Part II.
1.5 preparation of reagents
1.5.1PD98059(20mmol/L)
Take 5.3456mg PD98059, dissolve with 1mLDMSO, filter, 0.1mL subpackage ,-80 DEG C save backup.
1.5.2SP600125(20mmol/L)
Take 4.4046mg SP600125, dissolve with 1mLDMSO, filter, 0.1mL subpackage ,-80 DEG C save backup.
1.5.3SB203580(20mmol/L)
Take 7.5476mg SP600125, dissolve with 1mLDMSO, filter, 0.1mL subpackage ,-80 DEG C save backup.
All the other same Part II.
2 experimental techniques
2.1 cell survival rate
2.1.1 method
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in Part I 2.1.2 is 3 × 10
4the cell suspension of individual/mL, is inoculated on 96 orifice plates, is placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2after cultivating 24h in incubator, be divided into Normal group, H
2o
2damage group, Edaravone group (160 μ g/mL), 4-methoxyl group benzylalcohol (20 μ g/mL, 40 μ g/mL, 80 μ g/mL) three dosage groups and inhibitor group (normal+inhibitor, H
2o
2+ inhibitor, Eda-160+ inhibitor, MA-20+ inhibitor, MA-40+ inhibitor, MA-80+ inhibitor), often group establishes 6 multiple holes, respectively after administration 24h, inhibitor component does not add 50 μm of ol/L PD98059, SP60012, SB203580 and hatches 1h, except Normal group, all the other each group H all adding 50 μm of ol/L
2o
2modeling liquid, damage 2h, then careful suction abandons original fluid, slowly adds containing serum RPMI-1640 culture fluid, be placed in CO along hole wall edge
2continue in incubator to cultivate 42h, measure cell survival rate with mtt assay.
2.1.2 result
Result shows, and after adding ERK1/2 inhibitor PD98059, jnk inhibitor SP600125, p38 inhibitor SB203580, compare with each administration group, inhibitor group cell survival rate obviously reduces (P < 0.01), the results are shown in Table 9.
Table 9 cell survival rate (
n=6)
Note: compare with Control group and have statistical significance * * P<0.01, * P<0.05; Compare with Model group and have statistical significance
##p<0.01,
#p<0.05;
Compare with Eda-160 group and have statistical significance
＄ ＄p<0.01; Compare with MA-20 group and have statistical significance
▲ ▲p<0.01;
Compare with MA-40 group and have statistical significance
■ ■p<0.01; Compare with MA-80 group and have statistical significance
¤ ¤p<0.01
2.2 4-methoxyl group benzylalcohols are on the impact of ERK1/2, P-ERK1/2, JNK, P-JNK, p38, P-p38 protein expression
2.2.1 method
When PC12 Growth of Cells is to exponential phase, making cell concentration by method described in Part I 2.1.2 is 2 × 10
5the cell suspension of individual/mL, is inoculated on 6 orifice plates, is placed in 37 DEG C, 5%CO
2, 95% air, saturated humidity CO
2cultivate 24h in incubator, experiment grouping and administration the same, after modeling 2h, extract protein sample, carry out Western-blot analysis, the same Part II of associated operating steps.
Antibody dilution ratio
2.2.1 result
Compare with normal group, model group P-ERK1/2, P-JNK, P-p38 protein expression increase (P < 0.01), compare with model group, Edaravone 160 μ g/mL group P-ERK1/2, P-p38 protein expression increases (P < 0.01 or P < 0.05), and suppress p-JNK protein expression (P < 0.01), 4-methoxyl group benzylalcohol can increase P-ERK1/2 protein expression (P < 0.01 or P < 0.05), suppress P-JNK protein expression (P < 0.01), PD98059, SP600125, after SB203580 pretreatment, P-ERK1/2 in cell, P-JNK, the expression of P-p38 albumen is obviously suppressed (P < 0.01 or P < 0.05), between each group, ERK1/2, JNK, p38 protein expression does not have significant difference.The results are shown in Figure 3, table 10.
Table 10 4-methoxyl group benzylalcohol is to H
2o
2cause ERK1/2, JNK, p38 protein expression after PC12 cell oxidative damage impact (
n=3)
Note: compare with Control group and have statistical significance * * P<0.01, * P<0.05; Compare with Model group and have statistical significance
##p<0.01,
#p<0.01
Compare with Eda-160 group and have statistical significance
＄ ＄p<0.01; Compare with MA-20 group and have statistical significance
▲ ▲p<0.01;
Compare with MA-40 group and have statistical significance
■ ■p<0.01; Compare with MA-80 group and have statistical significance
¤ ¤p<0.01
Table 11 4-methoxyl group benzylalcohol is to H
2o
2cause P-ERK1/2, P-JNK, P-p38 protein expression after PC12 cell oxidative damage impact (
n=4)
Note: compare with Control group and have statistical significance * * P<0.01, * P<0.05; Compare with Model group and have statistical significance
##p<0.01,
#p<0.01
Compare with Eda-160 group and have statistical significance
＄ ＄p<0.01; Compare with MA-20 group and have statistical significance
▲ ▲p<0.01;
Compare with MA-40 group and have statistical significance
■ ■p<0.01; Compare with MA-80 group and have statistical significance
¤ ¤p<0.01
Discuss
PC12 cell is a kind of neuronal precursor system with differentiation potential, has the characteristic of sympathetic neuron sample, and has the advantage being easy to cultivate, and is widely used as the model of Studies On Neuronal.Therefore, we select PC12 cell as experimental subject, with H
2o
2process cell, copies oxidativestress damage neural cell model, for disclosing pharmaceutically-active Mechanism Study.
This experiment H
2o
2process PC12 cell 2h, MTT colorimetric method for determining cell survival rate, finds: growth and the propagation of cell all show as depression effect.
4-methoxyl group benzylalcohol 0.145mmol/L-0.58mmol/L (20 μ g/mL-80 μ g/mL) effectively can reduce the inhibitory action of oxidativestress damage to PC12 Growth of Cells and propagation, and in concentration dependent (table 1).
H
2o
2be the easy permeability strong oxidizer of a kind of film, it mainly by increasing ROS in cell, seriously disturbs Antioxidative Defense System function to the oxidativestress damage of cell, therefore measure intracellular ROS level can indirectly reflect cellular oxidation stress degree.The disequilibrium of oxidation and antioxidation of SOD and GSH to body plays vital effect, and their major physiological effect is the free radical can disposed in human body, is the antioxidant in body.
experimental result shows: 4-methoxyl group benzylalcohol can suppress H 2 o 2 intracellular ROS level after damage, and improve H 2 o 2 sOD activity and GSH in the rear cell of damage content, confirms that 4-methoxyl group benzylalcohol has protective effect to PC12 cellular oxidation stress damage further.
Caspase-3 is a key enzyme in apoptosis process, and its activation is the mark that apoptosis enters the irreversible stage.Caspase-3 is present in bag slurry with the form of proenzyme in normal state, does not have activity; But when apoptosis, it is activated, the Caspase-3 of activation is made up of two large subunits and small subunit, and cracking corresponding endochylema karyon substrate, finally causes apoptosis.And the decline of the MMP hallmark events that to be apoptosis early stage.H
2o
2process PC12 cell 2h, in cell, Caspase-3 is activated, and MMP declines, and PC12 apoptosis is described,
4-methoxyl group benzylalcohol can suppress caspase-3 activation and MMP decline, and prove that 4-methoxyl group benzylalcohol may protect PC12 cell by anti-apoptotic.
Apoptosis mainly contains two approach: mitochondria pathway and endoplasmic reticulum-induced.Caspase-9 and Caspase-12 is the important composition member of regulating cell apoptosis in Caspase family, and Caspase-9 is that the apoptosis of mitochondria pathway starts one of factor, and Caspase-12 is present in endoplasmic reticulum, is that er stress apoptosis is necessary.The activation of Caspase-9 and Caspase-12 finally all can activate Caspase-3 further, and trigger cell apoptosis.Protein expression result shows: 4-methoxyl group benzylalcohol can reduce the expression of Caspase-9, and then suppresses Caspase-3 activation, illustrates that the anti-apoptotic effect of 4-methoxyl group benzylalcohol mainly concentrates on mitochondria pathway.
MAPK is class serine/threonine protein kitase extracellular stimulus signal being converted to thin intramicellar reaction widely, the effect of important cellular signal transduction is played in many physiological process, comprise ERK1/2, JNK and p38 approach, it participates in regulation and control partial mitochondrial apoptosis.When CIR occurs, MAPK path is subject to the stimulation of ROS and is activated, the ERK1/2 activated can phosphorylation the expression of cyclin kinase inhibitors such as deactivation P27, P21, the propagation of irritation cell, simultaneously can the activation of apoptosis inhibit enzyme (as Caspases), and the expression of some anti-apoptosis factors can be induced, reduce apoptosis, reduce wound scope, reduce the impact that reperfusion injury brings body; JNK is after being subject to extracellular stimulus signal, be transferred near mitochondrial membrane, the Bcl-2 on mitochondrial membrane is suppressed after phosphorylation, cytochrome C is made to be released into endochylema, be combined with the caspase-9/ apoptotic proteins enzyme activition factor-1, final rise Caspase apoptosis pathway, inspires Caspase9-Caspase3 cascade reaction, causes apoptosis.Bax gene inflow line plastochondria can be stimulated after p38 is activated to cause cell death.Western-blot result display herein: in normal cell, phosphorylated CREB 1/2, JNK and p38MAPK albumen are expressed hardly, are subject to H
2o
2after stimulation, in highly expressing;
4-methoxyl group benzylalcohol has enhancing the effect that ERK1/2 phosphorylated protein is expressed, and phosphorylation JNK protein expression can be suppressed, illustrate that 4-methoxyl group benzylalcohol can anti-H is played by ERK1/2 and JNK signal path 2 o 2 the effect of induction PC12 cell injury.
Conclusion
1,4-methoxyl group benzylalcohol has anti-H
2o
2the effect of induction PC12 cell oxidative damage, its mechanism of action may be by suppressing endogenous mitochondrial apoptotic pathways.
2,4-methoxyl group benzylalcohol is by ERK1/2 and JNK signal path regulating cell apoptosis, to protect oxidative damage PC12 cell.
To sum up, 4-methoxyl group benzylalcohol is by the effect reducing intracellular ROS level, improve SOD vigor, improve GSH content and/or anti-apoptotic performance anti-oxidative damage, thus raising cell survival rate, can prepare and become antioxidant, for preventing anti-oxidative damage, prevent and/or treat oxidativestress damage, application prospect is good.
Claims (9)
1.4-methoxyl group benzylalcohol is preparing the purposes in antioxidant medicine.
2. purposes according to claim 1, is characterized in that: described antioxidant medicine is the medicine of anti-oxidative damage.
3. purposes according to claim 2, is characterized in that: the medicine of described anti-oxidative damage is the medicine preventing and/or treating oxidativestress damage.
4. the purposes according to claims 1 to 3 any one, is characterized in that: described medicine is the medicine reducing ROS level, improve SOD vigor, improve GSH content and/or anti-apoptotic.
5. purposes according to claim 4, is characterized in that: described preparation is oral formulations.
6. purposes according to claim 5, is characterized in that: described oral formulations is capsule, tablet.
7. a medicine for anti-oxidative damage, is characterized in that: it be with 4-methoxyl group benzylalcohol for active component, add the preparation that pharmaceutically acceptable adjuvant is prepared from.
8. purposes according to claim 7, is characterized in that: described preparation is oral formulations.
9. purposes according to claim 8, is characterized in that: described oral formulations is capsule, tablet.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101952233A (en) * | 2005-08-29 | 2011-01-19 | 沙伯基础创新塑料知识产权有限公司 | Method of making benzylated phenols |
| EP2669353A1 (en) * | 2011-01-28 | 2013-12-04 | Watanabe Oyster Laboratory Co., Ltd. | Antioxidant, antioxidant composition and production method therefor |
| CN103666767A (en) * | 2014-01-07 | 2014-03-26 | 新疆大学 | Preparation method of salix caprea volatile oil and application thereof |
-
2015
- 2015-05-27 CN CN201510278051.5A patent/CN104922096A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101952233A (en) * | 2005-08-29 | 2011-01-19 | 沙伯基础创新塑料知识产权有限公司 | Method of making benzylated phenols |
| EP2669353A1 (en) * | 2011-01-28 | 2013-12-04 | Watanabe Oyster Laboratory Co., Ltd. | Antioxidant, antioxidant composition and production method therefor |
| CN103666767A (en) * | 2014-01-07 | 2014-03-26 | 新疆大学 | Preparation method of salix caprea volatile oil and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BYEOUNG-SOO PARK等: "antioxidant activity and characterization of volatile constituents of Taheebo (Tabebuia impetiginosa Martius ex DC)", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| JESUS F. ARTEAGA等: "Comparison of the simple cyclic voltammetry (CV) and DPPH assays for the determination of antioxidant capacity of active principles", 《MOLECULES》 * |
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