CN110123882A - A kind of preparation method and application of SIRT1 activator - Google Patents
A kind of preparation method and application of SIRT1 activator Download PDFInfo
- Publication number
- CN110123882A CN110123882A CN201910374120.0A CN201910374120A CN110123882A CN 110123882 A CN110123882 A CN 110123882A CN 201910374120 A CN201910374120 A CN 201910374120A CN 110123882 A CN110123882 A CN 110123882A
- Authority
- CN
- China
- Prior art keywords
- preparation
- sirt1
- cell
- sirt1 activator
- rhizome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pulmonology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Organic Chemistry (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses the preparation methods and application of a kind of SIRT1 activator, the preparation of Rhizoma Fagopyri Dibotryis extract: it will be impregnated together after cymose buckwheat rhizome rhizome slice with clear water, water proof boiling 3h after being sealed, the medical fluid of acquisition is cooling, filtrate is filtered with 0.45 μm of filter membrane, filtrate is freeze-dried up to Rhizoma Fagopyri Dibotryis extract, the Rhizoma Fagopyri Dibotryis extract of freeze-drying is dissolved in dimethyl sulfoxide DMSO；It is added to culture medium and effectively facilitates SIRT1 protein expression, effectively mitigate the cytotoxicity of antimony chloride, the BEAS-2b cell viability for inhibiting antimony chloride to mediate loses and apoptosis.
Description
Technical field
The present invention relates to technical field of environmental science more particularly to a kind of preparation methods and application of SIRT1 activator.
Background technique
Cymose buckwheat rhizome derives from the dry rhizome of polygonaceae plant cymose buckwheat rhizome Fagopyrum dihotrys (D. Don) Hara,
It records in " Chinese Pharmacopoeia " 2015 version one, has effects that clearing heat and detoxicating, apocenosis dissolving stasis, be used for lung carbuncle pyemesis, lung heat asthma
The diseases such as cough.
Antimony (Antimony, Sb) is a kind of limited non-ferrous metal of resource, be widely used in rubber, ceramics, enamel,
Semiconductor original part, pigment, the fields such as plastics, medicine, glass, battery, paint, pyrotechnic material, alloy and fire retardant.China is
The most abundant country of antimony resource accounts for the 66.2% of world's gross reserves, and it is total that Chinese mine antimony yield (18.74 ten thousand tons) in 2010 accounts for the world
The 89.33% of yield, it means that China is just carry antimony ore exploitation and the processing serious environmental risk of bring.A large amount of cities
Waste and coal burning are the main sources for causing natural environment antimony pollution, and antimony is in air particle (main inducing of haze)
Major metal component；And a large amount of antimony are sucked and its compound is the main exposure of occupational environment in antimony ore exploitation and smelting process
Mode.Since respiratory tract exposure is the main route of exposure of antimony, pulmonary toxicity effect and Mechanism Study are antimony toxicologic studies
Key content, disclose the pulmonary toxicity mechanism of antimony and provide fundamental basis to intervene its injury of lungs.Article before me shows chlorination
Antimony can promote SIRT1 protein degradation, inhibit the genetic transcription of SIRT1, and SIRT1 protein expression is both caused to be lowered, from
And cause alveolar epithelial cells apoptosis.Gene means are overexpressed SIRT1 or (a kind of generally acknowledged SIRT1 is sharp using resveratrol
Agent living, the purchase of sigma company) SIRT1 can be promoted to express, inhibit the cytotoxicity of antimony chloride.
Find one and long-lived relevant gene in saccharomycete in Ivy in 1986 et al., referred to as silent message adjusting because
Sub- 2(Sirt2), coming years find the homologous protein of Sirt2 a series of in mammals successively, are named as SIRT1-
SIRT7, wherein the Sirt2 homology highest of SIRT1 and yeast.SIRT1 is widely distributed as intracellular important regulatory factor
In the endochylema and karyon of mankind's various kinds of cell, participates in adjusting inflammation, cell cycle, aging and apoptosis, important physiology mistake stress be waited
Journey.The study found that the expression downward of SIRT1 is related with the occurrence and development of a variety of pulmonary diseases.Resveratrol is one kind of SIRT1
Native agonist, it has important protective effect to various pulmonary, while being also most common SIRT1 agonist.
Summary of the invention
The present invention provides the preparation method and application of a kind of SIRT1 activator, solves current commercialization in the prior art
Resveratrol price it is relatively high, itself there are the technical problems such as certain cytotoxicity.
The invention adopts the following technical scheme: a kind of preparation method of SIRT1 activator, step are as follows:
The preparation of Rhizoma Fagopyri Dibotryis extract: the first step will impregnate together after cymose buckwheat rhizome rhizome slice with clear water, water proof after being sealed
Boiling 3-4 hours, the medical fluid of acquisition is cooling, filtrate is filtered with 0.45 μm of filter membrane, filtrate is freeze-dried and is mentioned up to cymose buckwheat rhizome
Take object；
Step 2: the Rhizoma Fagopyri Dibotryis extract of freeze-drying is dissolved in dimethyl sulfoxide, the storage mother liquor of cell processed material is obtained,
5-10 hydrotropy is blown and beaten in the EP Guan Liyong pipette tips of 1.5mL；
Step 3: the mother liquor of cell processed material is dispensed, then puts -20 degree refrigerators and save, obtain SIRT1 activator.
As a preferred technical solution of the present invention: cymose buckwheat rhizome rhizome slice and clear water impregnate specifically together in the first step
Condition is to take 1 kilogram of cymose buckwheat rhizome rhizome slice, adds clear water to cross powder, impregnates a night, second day, adds 5 times of immersion water volumes
Water proof boiling after watertight is sealed.
As a preferred technical solution of the present invention: water proof conditions of cooking is that first decoction half is small with high heat in the first step
When, then change mild fire and continue to heat, closes fire after maintaining 3 hours.
As a preferred technical solution of the present invention: the cooling precondition of a step Chinese medicine liquid is to naturally cool to room temperature.
As a preferred technical solution of the present invention: the specific preparation step of storage mother liquor of cell processed material in second step
It is added in cell super-clean bench in the DMSO of 1ml for the Rhizoma Fagopyri Dibotryis extract powder 10-200mg of freeze-drying, 1.5mL's
EP Guan Liyong pipette tips blow and beat 5-10 hydrotropy.
As a preferred technical solution of the present invention: the condition dispensed in third step is to be divided in PCR pipe, each
Pipe puts 50 μ L, is placed in -20 degree refrigerators and saves.
The present invention also provides application of the SIRT1 activator made from above-mentioned preparation method in activation SIRT1.
The utility model has the advantages that
The preparation method of SIRT1 activator of the present invention a kind of and application compared with the prior art by using the above technical solution,
It has following technical effect that Rhizoma Fagopyri Dibotryis extract can effectively facilitate SIRT1 protein expression, effectively mitigates the cell toxicant of antimony chloride
Property, inhibit cell viability loss and apoptosis, cymose buckwheat rhizome are a potential SIRT1 agonists.There is potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is after the application SIRT1 activator of CCK8 detection various concentration is handled BEAS-2b cell 24 hours to cell
The influence diagram of vigor.
Fig. 2 is the intervention effect figure that the application SIRT1 activator declines cell viability caused by antimony chloride.
Fig. 3 is that the application SIRT1 activator and (or) antimony chloride are handled BEAS-2b cell 24 hours, and optical microscopy is clapped
Take the photograph cellular morphology figure.
Fig. 4 is the intervention effect figure that Tunel method detects the application SIRT1 activator to Apoptosis caused by antimony chloride.
Fig. 5 is the application SIRT1 activator and (or) after antimony chloride acts on BEAS-2b cell 24 hours, immunoblotting
Detect SIRT1 protein expression figure.
Fig. 6 is the application SIRT1 activator and (or) after antimony chloride acts on BEAS-2b cell 24 hours, SIRT1 egg
White matter expression statistical chart.
Specific embodiment
Below in conjunction with example, the invention will be further described, and embodiment is only used for that the present invention will be described, not
Constitute limitation to scope of the claims, it may occur to persons skilled in the art that other alternative means, in right of the present invention
In claimed range.
Embodiment 1:
Step 1: the preparation of Rhizoma Fagopyri Dibotryis extract: 1 kilogram of cymose buckwheat rhizome rhizome slice is taken, adds clear water to cross powder, impregnates a night, the
Two days, add 5 times immersion water volumes watertights seal after water proof boiling, first decoct with high heat half an hour, then change mild fire after
Continuous heating closes fire after maintaining 3 hours, the medical fluid of acquisition is naturally cooled to room temperature, filtrate is filtered with 0.45 μm of filter membrane, by filtrate
It is freeze-dried up to Rhizoma Fagopyri Dibotryis extract.
Step 2: the Rhizoma Fagopyri Dibotryis extract powder 10mg of freeze-drying to be added to the DMSO of 1ml in cell super-clean bench
In, the storage mother liquor of cell processed material is obtained, blows and beats 5-10 hydrotropy in the EP Guan Liyong pipette tips of 1.5mL.
Step 3: the mother liquor of cell processed material is attached separately in PCR pipe, each pipe puts 50 μ L, then puts -20
It spends refrigerator to save, obtains SIRT1 activator, dissolve at room temperature, be added to cell culture medium.
Application of the SIRT1 activator made from above-mentioned preparation method in activation SIRT1, step are as follows:
Step 1: BEAS-2b cell routinely training method carry out sterile culture, culture medium condition DMEM/F12+10%FBS,
It is added after culture solution in 37 DEG C, 5%CO2Incubator in cultivate.Before experiment, the cell of different densities is inoculated into as requested
Required culture dish or culture plate, adherent 24 hours.
Step 2: taking out the cymose buckwheat rhizome of pipe freezing, room temperature melts, and operates in cell super-clean bench, is added to culture medium,
Isometric DMSO is added as solvent control in control group, meanwhile, antimony chloride exposure is carried out to cell.
Step 3: cell viability is detected using CCK8 after processing 24 hours, it is every by 10000 cells in 96 orifice plates
The density in hole is inoculated with BEAS-2b, and every pore volume is 100 μ L, and adherent 24 hours.
Step 4: configuring the fresh complete culture containing antimony chloride or cymose buckwheat rhizome extracting solution in sterile 1.5 mL EP pipe
Base carries out 96 orifice plates to change liquid processing, every hole or 100 μ L culture mediums, 6 repeated samples of each processing, it is small to continue culture 24
When.
It takes pictures step 5: first being observed under inverted microscope, carries out the subsequent liquid that changes after picture under acquisition light field and handle, completely
According to the proportional arrangement mixed liquor of 10:1, every 100 μ L of hole changes liquid processing for culture medium and CCK8 reaction solution, and 37 °C to be incubated for 2 small
When.
Step 6: absorbance value at multi-function microplate reader detection 450nm, the vigor size of OD value size expression cell, respectively
Group and the ratio of control group are versus cell vigor.
Embodiment 2:
Step 1: the preparation of Rhizoma Fagopyri Dibotryis extract: 1 kilogram of cymose buckwheat rhizome rhizome slice is taken, adds clear water to cross powder, impregnates a night, the
Two days, add 5 times immersion water volumes watertights seal after water proof boiling, first decoct with high heat half an hour, then change mild fire after
Continuous heating closes fire after maintaining 3 hours, the medical fluid of acquisition is naturally cooled to room temperature, filtrate is filtered with 0.45 μm of filter membrane, by filtrate
It is freeze-dried up to Rhizoma Fagopyri Dibotryis extract.
Step 2: the Rhizoma Fagopyri Dibotryis extract powder 50mg of freeze-drying to be added to the DMSO of 1ml in cell super-clean bench
In, the storage mother liquor of cell processed material is obtained, blows and beats 5-10 hydrotropy in the EP Guan Liyong pipette tips of 1.5mL.
Step 3: the mother liquor of cell processed material is attached separately in PCR pipe, each pipe puts 50 μ L, then puts -20
It spends refrigerator to save, obtains SIRT1 activator, dissolve at room temperature, be added to cell culture medium.
Application of the SIRT1 activator made from above-mentioned preparation method in activation SIRT1, step are as follows:
Step 1: BEAS-2b cell routinely training method carry out sterile culture, culture medium condition DMEM/F12+10%FBS,
It is added after culture solution in 37 DEG C, 5%CO2Incubator in cultivate.Before experiment, the cell of different densities is inoculated into as requested
Required culture dish or culture plate, adherent 24 hours.
Step 2: taking out the cymose buckwheat rhizome of pipe freezing, room temperature melts, and operates in cell super-clean bench, is added to culture medium,
Isometric DMSO is added as solvent control in control group, meanwhile, antimony chloride exposure is carried out to cell.
Step 3: dUTP Nick End labelling method Tunel detects Apoptosis, in 24 orifice plates after processing 24 hours
Sterile slide is added in face, is then inoculated with BEAS-2b according to the density of the every ware of 50,000 cells, and every pore volume is 0.5 mL, and adherent 24 is small
When.
Step 4: the fresh complete medium containing antimony chloride and cymose buckwheat rhizome extracting solution is configured in sterile 2 mL pipe, it is right
24 orifice plates carry out changing liquid processing, every hole or 0.5 mL culture medium, 3 repeated samples of each processing, continue culture 24 hours.
Step 5: PBS is cleaned 2 times, every time 2 minutes；0.5 mL, 4% paraformaldehyde is added in every hole, fixed in 4 degree of refrigerators
15 minutes, after fixed, PBS was cleaned 3 times, every time 5 minutes.
Step 6: the Triton X-100,4 DEG C of 15 min of rupture of membranes of 0.5 mL 0.5% is added in every hole；PBS is cleaned 3 times,
5 minutes every time.
Step 7: dripping the equilibrium liquid of upper 50 μ L on plastic seal membrana oralis, slide is pulled out with syringe needle, by slide
It tips upside down on equilibrium liquid and is incubated at room temperature 10 minutes.
Step 8: spreading sealed membrane in wet box, the TdT enzyme reaction solution of upper 50 μ L is dripped, by 44 μ L equilibrium liquids, 5 μ L cores
Thuja acid mixture, 1 μ L rTdT enzyme composition, tips upside down on reaction solution for slide, is protected from light in 37 °C 60 minutes.
Step 9: dripping upper 100 μ L, 2 × SSC terminate liquid on plastic seal membrana oralis, slide is faced up and is placed in 2 holes
Plate；Then PBS is cleaned 3 times, every time 5 minutes.
Step 10: 500 μ L, 0.5 μ g/mL DAPI is added in every hole is protected from light 10 min of effect, then PBS is cleaned 3 times,
5 minutes every time.
Step 11: 87% glycerol mounting, coverslip has cell one, and 4 DEG C are kept in dark place down.
Step 12: exciting Tunel with blue light with burst of ultraviolel DAPI, observation is carried out with fluorescence microscope and is taken pictures.
Embodiment 3:
Step 1: the preparation of Rhizoma Fagopyri Dibotryis extract: 1 kilogram of cymose buckwheat rhizome rhizome slice is taken, adds clear water to cross powder, impregnates a night, the
Two days, add 5 times immersion water volumes watertights seal after water proof boiling, first decoct with high heat half an hour, then change mild fire after
Continuous heating closes fire after maintaining 3 hours, the medical fluid of acquisition is naturally cooled to room temperature, filtrate is filtered with 0.45 μm of filter membrane, by filtrate
It is freeze-dried up to Rhizoma Fagopyri Dibotryis extract.
Step 2: the Rhizoma Fagopyri Dibotryis extract powder 100mg of freeze-drying to be added to the DMSO of 1ml in cell super-clean bench
In, the storage mother liquor of cell processed material is obtained, blows and beats 5-10 hydrotropy in the EP Guan Liyong pipette tips of 1.5mL.
Step 3: the mother liquor of cell processed material is attached separately in PCR pipe, each pipe puts 50 μ L, then puts -20
It spends refrigerator to save, obtains SIRT1 activator, dissolve at room temperature, be added to cell culture medium.
Application of the SIRT1 activator made from above-mentioned preparation method in activation SIRT1, step are as follows:
Step 1: BEAS-2b cell routinely training method carry out sterile culture, culture medium condition DMEM/F12+10%FBS,
It is added after culture solution in 37 DEG C, 5%CO2Incubator in cultivate.Before experiment, the cell of different densities is inoculated into as requested
Required culture dish or culture plate, adherent 24 hours.
Step 2: taking out the cymose buckwheat rhizome of pipe freezing, room temperature melts, and operates in cell super-clean bench, is added to culture medium,
Isometric DMSO is added as solvent control in control group, meanwhile, antimony chloride exposure is carried out to cell.
Step 3:, using immune-blotting method SIRT1 protein expression, being pressed in 100 mm wares after processing 24 hours
The density of 1800000 every wares of cell is inoculated with BEAS-2b, and every ware volume is 12 mL, and adherent 24 hours.
Step 4: being configured in sterile 50 mL centrifuge tube fresh containing certain concentration antimony chloride and cymose buckwheat rhizome extracting solution
Complete medium carries out big ware to change liquid processing, and every ware or 12 mL culture mediums, 3 repeated samples of each processing continue to train
It supports 24 hours.
Step 5: PBS is cleaned 2 times, every time 2 minutes；The 150 incomplete lysates of μ LRIPA are added, protease is added and inhibits
Agent cocktail(1:50) and defoaming agent PMSF(1:100) complete lysate is formed, it prepares in advance, is placed in ice face pre-cooling；With
Cell scraper is placed in 1.5 mL pipes and cracks 30 min on ice by the cell scraper of pre-cooling.
Step 6: ultrasonication, 30% power, ultrasound 4 seconds are stopped 2 seconds, are repeated 3 times.
Step 7: being centrifuged protein liquid in 4 DEG C of centrifuges, condition is 16000 g, is centrifuged 10 min.
Step 8: supernatant is collected, with BCA protein quantification kit measurement protein concentration.
It adjusts step 9: taking equal protein according to protein concentration and cannoing be used up full lysate RIPA to same volume, according to
Protein abundance selects identical applied sample amount, is added 5 × Loading Buffer, 95 °C of 5 min of denaturation, of short duration centrifugation, set on ice or-
80 °C of preservations.
Step 10: SDS-PAGE electrophoresis: preparing the SDS- of various concentration according to the molecular size range of detected albumen
PAGE glue installs electrophoresis system after the knot that is gelled, and runs SIRT1 with 8% glue.
Step 11: loading carries out protein electrophoresis, 100 V of glue, 120 V of separation gel is concentrated, until bromophenol blue is just gone to point
Stop when from glue bottom.
Step 12: glass plate is pried open, movement is light when sled, sled repeatedly that be on two sides gently, until
Sled goes glass plate, and removal concentration glue cuts glue according to the position of destination protein.
Step 13: transferring film: NC film, 200 mA transferring film, 2 h or 90 mA transferring films are stayed overnight, and notice that ice bag is added in transferring film slot,
Transferring film box is placed in ice chest.
Step 14: carefully removing the NC film for having turned upper albumen to the end of transferring film, it is simple clear to be placed on progress in TBST
It washes away except transferring film liquid.
Step 15: closing: preparing 3% BSA-TBST as confining liquid, room temperature closes 2 h.
Step 16: primary antibody prepare: by different antibodies recommend diluted concentration with antibody processed in 1x TBST (SIRT1,1:
1000；ACTB, 1:30000), the Sodium azide that 1:1000 is added prevents long bacterium.
Step 17: 4 °C of primary antibody overnight (overnight film second day take out after need on shaking table 30 min of ambient-temp-stable), knot
Primary antibody is recycled after beam.
Step 18: TBST washes 3 times, 5 min every time.
Step 19: secondary antibody is incubated for: with the fluorescence secondary antibody (1:1000) of confining liquid dilution respective sources, room temperature is protected from light shaking table
It is incubated for 1 h.
Step 20: be protected from light, TBST washes 3 times, every time 5 min.
21st step: carrying out fluorescent scanning with Odyssey imager infrared laser two tone image analysis system, utilizes
Odyssey software analyzes gray value, using the ratio of SIRT1 and ACTB(internal reference) as the expression figureofmerit of SIRT1.
22nd step: the ratio of statistics mapping, each group and control group is opposite SIRT1 horizontal.
Embodiment 4:
Step 1: the preparation of Rhizoma Fagopyri Dibotryis extract: 1 kilogram of cymose buckwheat rhizome rhizome slice is taken, adds clear water to cross powder, impregnates a night, the
Two days, add 5 times immersion water volumes watertights seal after water proof boiling, first decoct with high heat half an hour, then change mild fire after
Continuous heating closes fire after maintaining 3 hours, the medical fluid of acquisition is naturally cooled to room temperature, filtrate is filtered with 0.45 μm of filter membrane, by filtrate
It is freeze-dried up to Rhizoma Fagopyri Dibotryis extract.
Step 2: the Rhizoma Fagopyri Dibotryis extract powder 200mg of freeze-drying to be added to the DMSO of 1ml in cell super-clean bench
In, the storage mother liquor of cell processed material is obtained, blows and beats 5-10 hydrotropy in the EP Guan Liyong pipette tips of 1.5mL.
Step 3: the mother liquor of cell processed material is attached separately in PCR pipe, each pipe puts 50 μ L, then puts -20
It spends refrigerator to save, obtains SIRT1 activator, dissolve at room temperature, be added to cell culture medium.
Application of the SIRT1 activator made from above-mentioned preparation method in activation SIRT1, step are as follows:
Step 1: BEAS-2b cell routinely training method carry out sterile culture, culture medium condition DMEM/F12+10%FBS,
It is added after culture solution in 37 DEG C, 5%CO2Incubator in cultivate.Before experiment, the cell of different densities is inoculated into as requested
Required culture dish or culture plate, adherent 24 hours.
Step 2: taking out the cymose buckwheat rhizome of pipe freezing, room temperature melts, and operates in cell super-clean bench, is added to culture medium,
Isometric DMSO is added as solvent control in control group, meanwhile, antimony chloride exposure is carried out to cell.
Step 3: detecting cell viability using CCK8, Tunel detects Apoptosis, immunoblotting after processing 24 hours
SIRT1 protein expression is detected, is inoculated with BEAS-2b by the density of 10000 cell per wells in 96 orifice plates, every pore volume is 100
μ L, adherent 24 hours.
Step 4: configuring the fresh complete culture containing antimony chloride or cymose buckwheat rhizome extracting solution in sterile 1.5 mL EP pipe
Base carries out 96 orifice plates to change liquid processing, every hole or 100 μ L culture mediums, 6 repeated samples of each processing, it is small to continue culture 24
When.
It takes pictures step 5: first being observed under inverted microscope, carries out the subsequent liquid that changes after picture under acquisition light field and handle, completely
According to the proportional arrangement mixed liquor of 10:1, every 100 μ L of hole changes liquid processing for culture medium and CCK8 reaction solution, and 37 °C to be incubated for 2 small
When.
Step 6: absorbance value at multi-function microplate reader detection 450nm, the vigor size of OD value size expression cell, respectively
Group and the ratio of control group are versus cell vigor.
Cell viability is detected using CCK8,50 and 100 μ g/ml cymose buckwheat rhizomes itself do not influence cell viability, can be with
Cell viability caused by antimony chloride Sb is inhibited to decline.As shown in Figure 1, the cymose buckwheat rhizome of various concentration (12.5-200 μ g/mL) is extracted
On cell viability without influence after object processing BEAS-2b cell 24 hours, it is safe that SIRT1, which activates concentration,；Fig. 2 is also to use
CCK8 method detects cell viability, and individual antimony handles 20 μM of significant decrease cell viabilities, is then added 50 in antimony processing cell
μ g/mL or 100 μ g/mL(safe concentrations) cymose buckwheat rhizome intervened, cell viability can restore, * *P<0.01。
As shown in figure 3, handling BEAS-2b cell 24 hours as illustrated, optical microscopy shoots cellular morphology, antimony chloride
Optical microphotograph microscopic observation cell after exposure, floating cells, apoptotic cell increased significantly, and Rhizoma Fagopyri Dibotryis extract obviously inhibits this
A phenomenon；Apoptosis is detected using Tunel, has a large amount of Tunel positive cell in antimony chloride processing cell as the result is shown, and
After Rhizoma Fagopyri Dibotryis extract is intervened, a small amount of Tunel positive cell can only be seen, further support Fig. 2 result.
After cell receives respective handling, total protein of cell, immune-blotting method protein expression level are extracted.As shown in figure 5,
After BEAS-2b cell exposure 24 hours, immune-blotting method SIRT1 protein expression, antimony chloride exposure is lowered in BEAS-2b cell
SIRT1 protein expression level, in spite of antimony chloride is exposed to, cymose buckwheat rhizome processing can raise cell SIRT1 albumen table
Up to level, the statistical result of Fig. 6 shows that difference has statistical significance.These results suggest that cymose buckwheat rhizome is potential as one
SIRT1 activator, up-regulation SIRT1 expression, mitigates the cytotoxicity of antimony chloride, * *P<0.01。
Claims (7)
1. a kind of preparation method of SIRT1 activator, which is characterized in that step are as follows:
The preparation of Rhizoma Fagopyri Dibotryis extract: the first step will impregnate together after cymose buckwheat rhizome rhizome slice with clear water, water proof after being sealed
Boiling 3-4 hours, the medical fluid of acquisition is cooling, filtrate is filtered with 0.45 μm of filter membrane, filtrate is freeze-dried and is mentioned up to cymose buckwheat rhizome
Take object；
Step 2: the Rhizoma Fagopyri Dibotryis extract of freeze-drying is dissolved in dimethyl sulfoxide, the storage mother liquor of cell processed material is obtained,
5-10 hydrotropy is blown and beaten in the EP Guan Liyong pipette tips of 1.5mL；
Step 3: the mother liquor of cell processed material is dispensed, then puts -20 degree refrigerators and save, obtain SIRT1 activator.
2. a kind of preparation method of SIRT1 activator according to claim 1, it is characterised in that: cymose buckwheat rhizome in the first step
It is to take 1 kilogram of cymose buckwheat rhizome rhizome slice that rhizome slice and clear water impregnate actual conditions together, adds clear water to cross powder, impregnates a night,
Second day, water proof boiling after adding the watertight of 5 times of immersion water volumes to seal.
3. a kind of preparation method of SIRT1 activator according to claim 1, it is characterised in that: Steam by water bath in the first step
Condition is boiled first to decoct with high heat half an hour, then changes mild fire and continues to heat, closes fire after maintaining 3-4 hours.
4. a kind of preparation method of SIRT1 activator according to claim 1, it is characterised in that: first step Chinese medicine liquid cooling
But precondition is to naturally cool to room temperature.
5. a kind of preparation method of SIRT1 activator according to claim 1, it is characterised in that: in second step at cell
The storage specific preparation step of mother liquor of reason object is the Rhizoma Fagopyri Dibotryis extract powder 10-200mg of freeze-drying in cell super-clean bench
It is added in the DMSO of 1ml, blows and beats 5-10 hydrotropy in the EP Guan Liyong pipette tips of 1.5mL.
6. a kind of preparation method of SIRT1 activator according to claim 1, it is characterised in that: dispensed in third step
Condition is to be divided in PCR pipe, and each pipe puts 50 μ L, is placed in -20 degree refrigerators and saves.
7. application of the SIRT1 activator made from any preparation method of claim 1-6 in activation SIRT1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374120.0A CN110123882A (en) | 2019-05-07 | 2019-05-07 | A kind of preparation method and application of SIRT1 activator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374120.0A CN110123882A (en) | 2019-05-07 | 2019-05-07 | A kind of preparation method and application of SIRT1 activator |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110123882A true CN110123882A (en) | 2019-08-16 |
Family
ID=67576419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910374120.0A Pending CN110123882A (en) | 2019-05-07 | 2019-05-07 | A kind of preparation method and application of SIRT1 activator |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110123882A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104922700A (en) * | 2015-06-18 | 2015-09-23 | 卢中秋 | Experimental method for applying Sirt1-Nrf2 novel oxidation resisting pathway in oxidative damage treatment |
| CN106176989A (en) * | 2015-05-03 | 2016-12-07 | 刘宽明 | A kind of formula controlling lung abscess |
| CN107029063A (en) * | 2017-05-08 | 2017-08-11 | 浙江农林大学 | A kind of haze that reduces is to the Chinese medicine composition that is damaged caused by respiratory tract and lung |
| CN108619250A (en) * | 2018-07-06 | 2018-10-09 | 徐自升 | Cymose buckwheat rhizome is fresh to carry spray-dried granules medicine materical crude slice |
-
2019
- 2019-05-07 CN CN201910374120.0A patent/CN110123882A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106176989A (en) * | 2015-05-03 | 2016-12-07 | 刘宽明 | A kind of formula controlling lung abscess |
| CN104922700A (en) * | 2015-06-18 | 2015-09-23 | 卢中秋 | Experimental method for applying Sirt1-Nrf2 novel oxidation resisting pathway in oxidative damage treatment |
| CN107029063A (en) * | 2017-05-08 | 2017-08-11 | 浙江农林大学 | A kind of haze that reduces is to the Chinese medicine composition that is damaged caused by respiratory tract and lung |
| CN108619250A (en) * | 2018-07-06 | 2018-10-09 | 徐自升 | Cymose buckwheat rhizome is fresh to carry spray-dried granules medicine materical crude slice |
Non-Patent Citations (4)
| Title |
|---|
| 彭成等主编: "《中国临床药物大辞典 中药饮片卷》", 31 August 2018, 中国医药科技出版社 * |
| 董六一等: "金荞麦对克雷伯杆菌肺炎大鼠肺组织损伤的保护作用及其机制 ", 《中药材》 * |
| 郭立兵等: "茶儿茶素防治放射性肺损伤的实验研究 ", 《现代医院》 * |
| 顾言等: "清肺汤对急性呼吸窘迫综合征患者呼出气冷凝液和血清中内皮素-1、前列腺素E_2的影响 ", 《实用医学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jones et al. | Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture | |
| Gillespie et al. | Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins | |
| Kershenobich et al. | The relationship between the free pool of proline and collagen content in human liver cirrhosis | |
| JP6850360B2 (en) | Clinically usable cell cryopreservation solution | |
| KR20070090187A (en) | Use of flow-cytometric analysis to optimize cell banking strategies for cho cells | |
| Goodwin et al. | 3D tissue-like assemblies: A novel approach to investigate virus–cell interactions | |
| CN104990780A (en) | Dedicated centrifugal tube and method for manufacturing cell blocks | |
| CN110123882A (en) | A kind of preparation method and application of SIRT1 activator | |
| US20210095244A1 (en) | Methods and Compositions for Storing Bacteria | |
| CA2968548A1 (en) | System for the concomitant assessment of drug dissolution, absorption and permeation and methods of using the same | |
| Luo et al. | Detecting reactive oxygen species generation and stem cell proliferation in the Drosophila intestine | |
| CN111269883A (en) | Preparation method of highly mutant urine stem cells in mitochondrial mt.3243A & gtG mutant population | |
| Seligmann Jr et al. | Freeze drying and residual moisture | |
| Thomson et al. | Observations on cryopreservation of lymphocytes in chronic lymphocytic leukaemia and normal human lymphocytes | |
| TW202012620A (en) | Method for circulating tumor cells isolation | |
| RU2737321C1 (en) | Method of preserving bacteria in faecal microbiota and bacterial cultures grown on dense agarized nutrient or differential media in low temperature using composite medium for freezing | |
| CA2983373C (en) | In vitro preservation of therapeutic cells | |
| Mooijman et al. | Optimisation of the ISO-method on enumeration of somatic coliphages (draft ISO 10705-2) | |
| WO2022147917A1 (en) | Use of sarms compound containing ester-aromatic propionamide and metabolite thereof in preparation of anti-novel-coronavirus drug | |
| CN113957053A (en) | Method for screening traditional Chinese medicine active ingredients of anti-intestinal cancer stem cells by using human intestinal cancer organoid | |
| Nichols Jr | Solubility of carbon dioxide in body fat | |
| CN110452948A (en) | Deer heart protein hydrolysate and its preparation method and application | |
| WO2019234288A1 (en) | Catching and enriching cultivation of microorganisms in solidifying density gradients | |
| Shier et al. | Hepatitis c virus genotype 4 replication in the hepatocellular carcinoma cell line HepG2/C3A | |
| CN108845042A (en) | The measuring method of 17 kinds of amino acid contents in yak meat |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190816 |