CN108330156A - A kind of big white orchid growing point extract safety detecting method - Google Patents
A kind of big white orchid growing point extract safety detecting method Download PDFInfo
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- CN108330156A CN108330156A CN201810253700.XA CN201810253700A CN108330156A CN 108330156 A CN108330156 A CN 108330156A CN 201810253700 A CN201810253700 A CN 201810253700A CN 108330156 A CN108330156 A CN 108330156A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Abstract
The invention discloses a kind of big white orchid growing point extract safety detecting methods, are related to technical field.The present invention includes SS01, detection cell survival degree：SS02, the protection of the orchid growing point extract DNA damage caused by ultraviolet light and due to oxidative damage is assessed：SS03, assessment orchid growing point extract maintenance Skin Cell caused by ultraviolet light damage.After the present invention to orchid extract by being detected experiment, new series cosmetics are developed as the additive of cosmetics using orchid extract liquor orchid growing point extract, improve the surcharge of orchid industry.
Description
Technical field
The invention belongs to extract safety detection technical fields, more particularly to a kind of big white orchid growing point extract
Safety detecting method.
Background technology
Cosmetics refer to interspersing among any position of human body surface, such as skin with smearing, sprinkling or other similar approach
Skin, hair refer to toenail, lips and teeth etc., to reach cleaning, maintenance, beauty, modification and change appearance, or correct human scent, protect
Hold the chemical industrial product or fine chemical product for the purpose of kilter.Makeup kind is mainly made of some additives；And
Some existing additives have skin certain detrimental effect；After 2010, zero burden product starts to be born, and a batch zero is negative
Product is carried on a shoulder pole, chemical composition unnecessary is reduced by leading, increases pure skin care ingredient and be the theme, give the female of used frequent cosmetics
Property the completely new change of friend took, " zero burden " product is mainly characterized by, and product fierceness reduces many unwanted contributions, shield
Skin ingredient, such as sodium hyaluronate, collagen etc. are active use, and direct percutaneous absorption, properties of product are extremely mild, even again
As long as fragile skin, using appropriate, generally also there is no problem, and therefore, this can make up and not destroy skin to greatest extent.
There are not the cosmetics of exploitation orchid growing point extract currently on the market；Therefore for using orchid extract liquor as change
It needs to be measured the damage of Skin Cell orchid before the cosmetics of the additive manufacture orchid series of cosmetic, the present invention one
The big white orchid growing point extract safety detecting method of kind.
Invention content
The purpose of the present invention is to provide a kind of big white orchid growing point extract safety detecting methods, are deposited by cell
Activity is tested；Cell cycle tests；Protection DNA damage caused by ultraviolet light and due to oxidative damage is tested；Maintenance is because of ultraviolet light
And oxidative damage and caused by Skin Cell damage test the safety of orchid extract is evaluated, solve existing orchid
The problem of serial cosmetics vacancy.
In order to solve the above technical problems, the present invention is achieved by the following technical solutions：
The present invention is a kind of big white orchid growing point extract safety detecting method, is comprised the following processes：
SS01, detection cell survival degree：
Specifically include whether assessment orchid growing point extract liquor has cytotoxicity, assessment orchid growing point extraction to Skin Cell
It takes liquid to change Skin Cell kenel, assesses the Skin Cell period；
SS02, the protection of the orchid growing point extract DNA damage caused by ultraviolet light and due to oxidative damage is assessed：
Specifically include orchid growing point extract protection cyclic DNA effective matrix, orchid growing point extract protected fragment
Change DNA effective matrix；
SS03, assessment orchid growing point extract maintenance Skin Cell caused by ultraviolet light damage：
Specifically include assessment extract maintenance Skin Cell cell survival degree, assessment detecting DNA damages after action of ultraviolet radiation
The ring fourth pyrimidine dimer production quantity of wound.
Further, in the SS01 detect cell survival degree in assessment orchid growing point extract liquor to Skin Cell whether
Have the process following steps of cytotoxicity：
S011, Skin Cell culture：Take human skin cutin strain cell culture in 96 well culture plates, and 96 holes are cultivated
Plate is placed on 37 DEG C and 5%CO2It is cultivated in incubator at least 24 hours；
S012, extract liquor configuration：Take the extraction for refrigerating spare orchid growing point extract and being configured to various concentration gradient
Take liquid；
S013, Cytotoxicity evaluation：The extract liquor of the various concentration of S012 configurations is added in 96 well culture plates into S011,
Culture solution is removed after reaction, and culture solution and the MTT solution reactions of 10ul are rejoined after being used in combination sterile PBS cleanings primary,
37 DEG C and 5%CO2Culture reaction removes culture solution after 4 hours in incubator, and the DMSO Rong Xie Jia Za precipitation of 100 μ L is added
Object finally measures light absorption value under wavelength 570nm.
Further, it is detected in the SS01 in cell survival degree and assesses orchid growing point extract liquor to Skin Cell kenel
The process following steps of variation：
S014 takes human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates be placed on 37 DEG C and
5%CO2It is cultivated in incubator at least 24 hours；
The extract of 1 μ L is added in 96 well culture plates of the S015 into S014, after the reaction of different time gradient, in microscope
Lower observation cellular morphology simultaneously photographs to record.
Further, the process following steps that the Skin Cell period is assessed in cell survival degree are detected in the SS01：
S021, human skin cutin strain cell culture is taken to be cultivated in 24 well culture plates at least 24 hours；
S022,10 μ L orchid growing point extracts are added, are reacted in incubator；
Supernatant and cell are taken out after S023, reaction, and are put into centrifuge tube and are centrifuged 5 minutes with 1200rpm；Remove supernatant
Liquid is added the PBS of 300 μ L, slowly shakes and moved in microcentrifugal tube after 700 μ L alcohol fixation cells are added dropwise, be placed in 4
DEG C refrigerator storage；
S024, microcentrifugal tube is centrifuged 5 minutes with 1200rpm, removes supernatant, the PBS of 445 μ L, 5 μ L is sequentially added
The Triton X-100 of the ribonuclease A of a concentration of 10mg/ml and 50 μ L a concentration of 10% reacts 30 in 37 DEG C
Minute the RNA of human skin cutin strain cell is destroyed after decomposing, after centrifuging 5 minutes with 1200rpm and remove supernatant, is added
The PBS for entering 400 μ L adds the polyimide solution of 5 a concentration of 5mg/ml of μ L and is protected from light at a temperature of 4 DEG C instead after mixing
It answers 5 minutes, to filter membrane filtration；
S025, using stream type cell analyzer and coordinate Winmdi softwares analyze cell cycle distribution ratio.
Further, cell density of the human skin cutin strain cell in 96 well culture plates is 1 × 104/
well。
Further, the process following steps for assessing orchid growing point extract protection cyclic DNA effect in SS02：
S0311, by pUC119DNA plasmids with 1:8 ratios are diluted with PBS；
S0312, the dilution in S0311 is taken to set up control group, ultraviolet light and hydrogen peroxide effect group, ultraviolet light and double respectively
Oxygen water effect+extract group；
S0313, it respectively takes pUC119DNA plasmid dilutions of the 2 μ L through S0312 step process to be placed in microcentrifugal tube, sees
It examines and judges whether extract has protection skin histology uvioresistant and oxidative damage efficiency；
Wherein, the extract in the ultraviolet light and hydrogen peroxide effect+extract group is matched using orchid growing point extract
At various concentration gradient extract liquor, in 37 DEG C act on 1 hour, be added load dyestuff mixing after, in the agar of 0.8% concentration
Electrophoresis is carried out in sugar juice, the percentage of S types DNA and L-type DNA are analyzed in latter electrophoresis film image acquisition system analysis in 30 minutes
Ratio.
Further, the process of orchid growing point extract protected fragment DNA effects walks as follows in the assessment SS02
Suddenly：
S0321, the DNA fragmentation of diluted 2 μ L standard molecular weights is added in micro tube；
S0322, control group, ultraviolet light and hydrogen peroxide effect group, ultraviolet light and hydrogen peroxide effect+extract are set up respectively
Group；
S0323, the orchid growing point extract liquor for taking various concentration gradient act on 1 hour in 37 DEG C, and it is mixed that load dyestuff is added
After conjunction, electrophoresis is carried out in the agarose solution of 0.8% concentration, latter electrophoresis film image acquisition system analysis in 30 minutes divides
Analyse the percentage of S types DNA and L-type DNA.
Further, the mistake of extract maintenance Skin Cell cell survival degree after action of ultraviolet radiation is assessed in the SS03
Journey following steps：
S0411, take human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates be placed on 37 DEG C with
And 5%CO2It is cultivated in incubator at least 24 hours；
S0412, control group, ultraviolet light irradiation group, ultraviolet light irradiation+extract processing group are set up respectively；S0413, reaction
After, remove culture solution, new culture solution and the MTT solution reactions of 10ul are cleaned and rejoined with PBS, 37 DEG C with
And 5%CO2Culture reaction removes culture solution after 4 hours in incubator, the DMSO Rong Xie Jia Za sediments of 100 μ L is added, finally
Light absorption value is measured under wavelength 570nm, the cell survival degree in each group is analyzed.
Further, the process of the ring fourth pyrimidine dimer production quantity of assessment detecting DNA damage walks as follows in the SS03
Suddenly：
S0421, cell density is taken to be 1 × 105The human skin cutin strain cell culture of/well is in 24 well culture plates
Culture at least 24 hours；
S0422, control group, ultraviolet light irradiation group, ultraviolet light irradiation+extract processing group are set up respectively；
S0423, treated cell is added in serum-free fresh medium and continues culture 4 hours, remove culture
Liquid is cleaned with PBS and ice methanol is added and fix cell, then acted on Triton X-100, and a concentration of 2mol/l is added
Hydrochloric acid act on 1 hour after space between cells is filled up with 1% bovine serum albumin(BSA) again, add CPD Primary antibodies in 37 DEG C of items
Reaction 1 hour is rocked under part, is taken out CPD Primary antibodies, is added CPD secondary antibodies and be protected from light 30 minutes, cleaned with PBS,
Detecting fluorescence performance under conditions of wavelength is 504-524nm；It adds fluorescent dye and carries out nuclear targeting, then with PBS
Detecting fluorescence performance under conditions of wavelength is 355-460nm after cleaning, and take pictures under the microscope in fluorescence microscopy；
S0424, detecting fluorescence performance under conditions of wavelength is 504-524nm to cell in each group, are 355- in wavelength
The photo detected under conditions of 460nm under fluorescence performance and microscope compares and analyzes.
Further, the control group is to remove cell supernatant, replaces serum fresh medium, after cultivating 4 hours, is used
It is placed in again in serum-free fresh medium after PBS cleanings and continues culture 4 hours；
Wherein, the ultraviolet light irradiation group is to remove cell supernatant, replaces serum fresh medium, after cultivating 4 hours,
It is cleaned and is drained with PBS after removing supernatant, after carrying out ultraviolet light irradiation respectively, serum-free fresh medium relaying is added immediately
Continuous culture 4 hours；
Wherein, the ultraviolet light irradiation+extract processing group is to remove cell supernatant, replaces serum fresh medium,
It after culture 4 hours, is cleaned and is drained with PBS after removing supernatant, after carrying out ultraviolet light irradiation respectively, be added contain extract immediately
Serum-free fresh medium in continue culture 4 hours.
The invention has the advantages that：
The present invention is tested by cell survival degree；Cell cycle tests；Protection is caused by ultraviolet light and due to oxidative damage
DNA damage is tested；The maintenance safety of Skin Cell damage test to orchid extract caused by due to ultraviolet light and oxidative damage
Property is evaluated；After the present invention is detected experiment to orchid extract, made using orchid extract liquor orchid growing point extract
New series cosmetics are developed for the additive of cosmetics, improve the surcharge of orchid industry.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Specific implementation mode
A kind of big white orchid growing point extract safety detecting method, comprises the following processes：
SS01, detection cell survival degree：
It is detected using MTT assay.By human skin cutin strain cell (1 × 104/ well) it cultivates in 96-well disks,
And at 37 DEG C and 5%CO2It is cultivated in incubator at least 24 hours.Whether assessment orchid growing point extract liquor has carefully Skin Cell
Cellular toxicity：The extract of various concentration is added and in specified time effect, reaches the reaction time, removes old culture solution, with
PBS cleanings are primary, and change new culture solution, and the MTT (3- (4,5-cimethylthiazol-2-yl) -2,5- of 10 μ L are added
Diphenyl tetrazolium bromide) solution reaction, in 37 DEG C, 5%CO2Reaction removes culture solution after 4 hours, is added
The DMSO of 100 μ L dissolves formazan sediments, and light absorption value (BioTek, Synergy are finally measured under wavelength 570nmTM2,
USA)。
Whether assessment orchid growing point extract liquor has Skin Cell the process following steps of cytotoxicity：
S011, Skin Cell culture：Take human skin cutin strain cell culture in 96 well culture plates, and 96 holes are cultivated
Plate is placed on 37 DEG C and 5%CO2It is cultivated in incubator at least 24 hours；
S012, extract liquor configuration：Take the concentration ladder for refrigerating spare orchid growing point extract and being configured to 1-20 ratios
The extract liquor of degree；
S013, Cytotoxicity evaluation：The extract liquor of the various concentration of S012 configurations is added in 96 well culture plates into S011,
Culture solution is removed after reaction, and culture solution and the MTT solution reactions of 10ul are rejoined after being used in combination sterile PBS cleanings primary,
37 DEG C and 5%CO2Culture reaction removes culture solution after 4 hours in incubator, and the DMSO Rong Xie Jia Za precipitation of 100 μ L is added
Object finally measures light absorption value under wavelength 570nm.
The process following steps that assessment orchid growing point extract liquor changes Skin Cell kenel：
Whether can change the kenel of Skin Cell using micro- sem observation orchid growing point extract liquor.By cell (1 × 104/
Well it) cultivates in 96-well disks, and at 37 DEG C and 5%CO2It is cultivated in incubator at least 24 hours.Be added the extract of 1 μ L with
And in specified time effect, after reaching the reaction time, cell kenel is observed under microscope (Nikon, TE2000-U, Japan),
And it photographs to record.
S014 takes human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates be placed on 37 DEG C and
5%CO2It is cultivated in incubator at least 24 hours；
The extract of 1 μ L is added in 96 well culture plates of the S015 into S014,5min, 10min, 15min, 20min,
After the time gradient reaction of 25min, in microscopically observation cellular morphology and photograph to record.
Assess the process following steps in Skin Cell period：
By 1 × 10410 μ L orchid growing points are added at least 24 hours in 24well disks in the culture of/well cell concentrations later
Extract reacts in incubator.Supernatant is collected later to take cell to 15mL centrifuge tubes, then with trypsin-EDTA solution
In centrifuge tube, centrifuged together with supernatant 1200rpm, 5 minutes.Supernatant is removed, 300 μ L PBS are added, slowly shake,
And 700 μ L dehydrated alcohols are added dropwise and fix cell, microcentrifugal tube is then moved to, is placed in 4 DEG C of refrigerators and stores.Flow cytometer
Before upper machine, cell is centrifuged 5 minutes at 4 DEG C with 1200rpm, removes supernatant, 445 μ L PBS, 5 μ L RNase are sequentially added
The RNA of cell is destroyed after decomposing, is reacted 30 minutes in 37 DEG C by (10mg/mL), 50 μ L 10%Triton X-100, with
1200rpm is centrifuged 5 minutes, removes supernatant, and 400 μ L PBS are added and are uniformly mixed, 5 μ L PI (5mg/mL) are added, in 4 DEG C
It is protected from light 5 minutes, to filter membrane filtration.Utilize stream type cell analyzer (flow cytometer, FACScan), cooperation
Winmdi computer softwares analyze cell cycle distribution ratio (%).
S021, human skin cutin strain cell culture is taken to be cultivated in 24 well culture plates at least 24 hours；
S022,10 μ L orchid growing point extracts are added, are reacted in incubator；
Supernatant and cell are taken out after S023, reaction, and are put into centrifuge tube and are centrifuged 5 minutes with 1200rpm；Remove supernatant
Liquid is added the PBS of 300 μ L, slowly shakes and moved in microcentrifugal tube after 700 μ L alcohol fixation cells are added dropwise, be placed in 4
DEG C refrigerator storage；
S024, microcentrifugal tube is centrifuged 5 minutes with 1200rpm, removes supernatant, the PBS of 445 μ L, 5 μ L is sequentially added
The Triton X-100 of the ribonuclease A of a concentration of 10mg/ml and 50 μ L a concentration of 10% reacts 30 in 37 DEG C
Minute the RNA of human skin cutin strain cell is destroyed after decomposing, after centrifuging 5 minutes with 1200rpm and remove supernatant, is added
The PBS for entering 400 μ L adds the polyimide solution of 5 a concentration of 5mg/ml of μ L and is protected from light at a temperature of 4 DEG C instead after mixing
It answers 5 minutes, to filter membrane filtration；
S025, using stream type cell analyzer and coordinate Winmdi softwares analyze cell cycle distribution ratio.
SS02, the protection of the orchid growing point extract DNA damage caused by ultraviolet light and due to oxidative damage is assessed：
Assess the process following steps of orchid growing point extract protection cyclic DNA effect：
By pUC119DNA plasmid with 1:8 ratios are diluted with PBS, distinctly handle -1. .Control groups, 2.
.UV with H2O2Effect group, 3. .UV and H2O2Effect+extract group.The out of the ordinary pUC119DNA plasmid for taking 2 μ L in it is micro from
After heart pipe, inquire into whether extract has protection Skin Cell uvioresistant and oxidative damage efficiency group：Take the extraction of various concentration
Object and UV+H2O2, it is acted on 1 hour in 37 DEG C, after loading dye mixing is added, electrophoresis is carried out in 0.8%agarose, 30 points
It is analyzed with electrophoresis film image acquisition system after clock, the percentage of analysis S-form DNA and L-form DNA.
S0311, by pUC119DNA plasmids with 1:8 ratios are diluted with PBS；
S0312, the dilution in S0311 is taken to set up control group, ultraviolet light and hydrogen peroxide effect group, ultraviolet light and double respectively
Oxygen water effect+extract group；
S0313, it respectively takes pUC119DNA plasmid dilutions of the 2 μ L through S0312 step process to be placed in microcentrifugal tube, sees
It examines and judges whether extract has protection skin histology uvioresistant and oxidative damage efficiency；
Wherein, the extract in the ultraviolet light and hydrogen peroxide effect+extract group is matched using orchid growing point extract
At various concentration gradient extract liquor, in 37 DEG C act on 1 hour, be added load dyestuff mixing after, in the agar of 0.8% concentration
Electrophoresis is carried out in sugar juice, the percentage of S types DNA and L-type DNA are analyzed in latter electrophoresis film image acquisition system analysis in 30 minutes
Ratio.
Assess the process following steps of orchid growing point extract protected fragment DNA effects：
0.5mL micro tubes are added in 2 μ L DNA Mw Standard Marker of dilution, distinctly handle -1.
.Control group, 2. .UV and H2O2Effect group, 3. .UV and H2O2Effect+extract group.Take the extract and UV+ of various concentration
H2O2, acted on 1 hour in 37 DEG C, loading dye mixing be added, with agaropectin gel nucleic acid electrophoretic analysis.
S0321, the DNA fragmentation of diluted 2 μ L standard molecular weights is added in micro tube；
S0322, control group, ultraviolet light and hydrogen peroxide effect group, ultraviolet light and hydrogen peroxide effect+extract are set up respectively
Group；
S0323, the orchid growing point extract liquor for taking various concentration gradient act on 1 hour in 37 DEG C, and it is mixed that load dyestuff is added
After conjunction, electrophoresis is carried out in the agarose solution of 0.8% concentration, latter electrophoresis film image acquisition system analysis in 30 minutes divides
Analyse the percentage of S types DNA and L-type DNA.
SS03, assessment orchid growing point extract maintenance Skin Cell caused by ultraviolet light damage：
Due to product using at first contact skin cuticula, this plan selection human skin cutin strain cell into
Row safety evaluation.It is detected using MTT assay.By skin keratinocytes (1 × 104/ well) it cultivates in 96-well disks, and
At 37 DEG C and 5%CO2It is cultivated in incubator at least 24 hours.
1.Control groups：Cell supernatant is removed, the fresh medium of serum-free is replaced, it is clear with PBS after cultivating 4 hours
The fresh medium for washing merging serum-free continues culture 4 hours, carries out depositing activity analysis.
2.UV irradiation groups：Cell supernatant is removed, the fresh medium of serum-free is replaced, places and is cultivated in cell incubator
It after 4 hours, removes supernatant and cleans and drain with PBS, after carrying out UV irradiations, the fresh medium of serum-free is added immediately, after
Continuous culture 4 hours, carries out depositing activity analysis.
3.UV irradiations+extract group：Extract is distinctly mixed in the fresh training of serum-free by the supernatant for removing cell
Nutrient solution after cultivating 4 hours, removes supernatant and is cleaned and drained with PBS, after progress UV irradiations out of the ordinary, is added immediately containing extract
Serum-free fresh medium, continue culture 4 hours, carry out depositing activity analysis.
4 reach the reaction time, remove old culture solution, primary with PBS cleanings, and change new culture solution, are added 10 μ L's
MTT (3- (4,5-cimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) solution reaction, in
37 DEG C, 5%CO2Reaction removes culture solution after 4 hours, the DMSO that 100 μ L are added dissolves formazan sediments, finally in wavelength
Light absorption value (BioTek, Synergy are measured under 570nmTM2,USA)。
The reason of excessive ultraviolet light irradiation causes DNA damage is that it can induce the DNA pyrimidine base bases adjacent in chain
Ring fourth pyrimidine dimer (CPD) photoproduct is generated, DNA space structures is made to change, to hinder DNA replication dna, transcription in turn
Influence the biological function of protein.Therefore, ultraviolet light irradiation causes the reparation of DNA damage to seem most important.By 1 × 105/mL
Cell culture in 24 porose discs at least 24 hours distinctly handle -1. .Control groups, 2. .UV irradiation groups, 3. .UV irradiations+
Extract group.Extract carries out UV irradiations after acting on 4 hours, replaces not serum-containing medium culture 4 hours.Remove culture solution with
PBS is cleaned, and fixes cell with ice methanol, then act on Triton X-100,2M HCl are added and act on 1 hour, then with 1%BSA
Space between cells is filled up, then rocks reaction 1 hour in 37 DEG C with CPD Primary antibodies, after removing Primary antibodies, adds secondary antibody
It is protected from light 30 minutes, is cleaned in Ex with PBS:504nm；Em:524nm detects fluorescence performance.Add Hoechst 33342
(10mg/mL) carries out nuclear targeting, again with Ex after being cleaned with PBS:355nm；Em:460nm detects fluorescence performance, and in fluorescence
Microscopically observation is taken pictures.
Assess the process following steps of extract maintenance Skin Cell cell survival degree after action of ultraviolet radiation：
S0411, take human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates be placed on 37 DEG C with
And 5%CO2It is cultivated in incubator at least 24 hours；
S0412, control group, ultraviolet light irradiation group, ultraviolet light irradiation+extract processing group are set up respectively；S0413, reaction
After, remove culture solution, new culture solution and the MTT solution reactions of 10ul are cleaned and rejoined with PBS, 37 DEG C with
And 5%CO2Culture reaction removes culture solution after 4 hours in incubator, the DMSO Rong Xie Jia Za sediments of 100 μ L is added, finally
Light absorption value is measured under wavelength 570nm, the cell survival degree in each group is analyzed.
Wherein, cell density of the human skin cutin strain cell in 96 well culture plates is 1 × 104/well。
Wherein, the process following steps of the ring fourth pyrimidine dimer production quantity of detecting DNA damage are assessed in SS03：
S0421, cell density is taken to be 1 × 105The human skin cutin strain cell culture of/well is in 24 well culture plates
Culture at least 24 hours；
S0422, control group, ultraviolet light irradiation group, ultraviolet light irradiation+extract processing group are set up respectively；
S0423, treated cell is added in serum-free fresh medium and continues culture 4 hours, remove culture
Liquid is cleaned with PBS and ice methanol is added and fix cell, then acted on Triton X-100, and a concentration of 2mol/l is added
Hydrochloric acid act on 1 hour after space between cells is filled up with 1% bovine serum albumin(BSA) again, add CPD Primary antibodies in 37 DEG C of items
Reaction 1 hour is rocked under part, is taken out CPD Primary antibodies, is added CPD secondary antibodies and be protected from light 30 minutes, cleaned with PBS,
Detecting fluorescence performance under conditions of wavelength is 504-524nm；It adds fluorescent dye and carries out nuclear targeting, then with PBS
Detecting fluorescence performance under conditions of wavelength is 355-460nm after cleaning, and take pictures under the microscope in fluorescence microscopy；
S0424, detecting fluorescence performance under conditions of wavelength is 504-524nm to cell in each group, are 355- in wavelength
The photo detected under conditions of 460nm under fluorescence performance and microscope compares and analyzes.
Wherein, control group is to remove cell supernatant, replaces serum fresh medium, after cultivating 4 hours, is cleaned with PBS
It is placed in again afterwards in serum-free fresh medium and continues culture 4 hours；
Wherein, the ultraviolet light irradiation group is to remove cell supernatant, replaces serum fresh medium, after cultivating 4 hours,
It is cleaned and is drained with PBS after removing supernatant, after carrying out ultraviolet light irradiation respectively, serum-free fresh medium relaying is added immediately
Continuous culture 4 hours；
Wherein, the ultraviolet light irradiation+extract processing group is to remove cell supernatant, replaces serum fresh medium,
It after culture 4 hours, is cleaned and is drained with PBS after removing supernatant, after carrying out ultraviolet light irradiation respectively, be added contain extract immediately
Serum-free fresh medium in continue culture 4 hours.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.There is no detailed for preferred embodiment
All details are described, are not limited the invention to the specific embodiments described.Obviously, according to the content of this specification,
It can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is in order to preferably explain the present invention
Principle and practical application, to enable skilled artisan to be best understood by and utilize the present invention.The present invention is only
It is limited by claims and its full scope and equivalent.
Claims (10)
1. a kind of big white orchid growing point extract safety detecting method, it is characterised in that：It comprises the following processes：
SS01, detection cell survival degree：
It specifically includes whether assessment orchid growing point extract liquor has cytotoxicity to Skin Cell, assesses orchid growing point extract liquor
Skin Cell kenel is changed, the Skin Cell period is assessed；
SS02, the protection of the orchid growing point extract DNA damage caused by ultraviolet light and due to oxidative damage is assessed：
Specifically include orchid growing point extract protection cyclic DNA effective matrix, orchid growing point extract protected fragment DNA
Effective matrix；
SS03, assessment orchid growing point extract maintenance Skin Cell caused by ultraviolet light damage：
Specifically include assessment extract maintenance Skin Cell after action of ultraviolet radiation cell survival degree, assessment detecting DNA damage it
Ring fourth pyrimidine dimer production quantity.
2. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
It states and detects whether assessment orchid growing point extract liquor in cell survival degree has the process of cytotoxicity such as to Skin Cell in SS01
Lower step：
S011, Skin Cell culture：Take human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates are put
At 37 DEG C and 5%CO2It is cultivated in incubator at least 24 hours；
S012, extract liquor configuration：Take the extract liquor for refrigerating spare orchid growing point extract and being configured to various concentration gradient；
S013, Cytotoxicity evaluation：The extract liquor of the various concentration of S012 configurations, reaction is added in 96 well culture plates into S011
After remove culture solution, culture solution and the MTT solution reactions of 10ul are rejoined after being used in combination sterile PBS cleaning primary, at 37 DEG C
And 5%CO2Culture reaction removes culture solution after 4 hours in incubator, the DMSO Rong Xie Jia Za sediments of 100 μ L is added, most
Afterwards light absorption value is measured under wavelength 570nm.
3. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
State the process following steps for detecting that assessment orchid growing point extract liquor changes Skin Cell kenel in cell survival degree in SS01：
S014 takes human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates are placed on 37 DEG C and 5%
CO2It is cultivated in incubator at least 24 hours；
The extract of 1 μ L is added in 96 well culture plates of the S015 into S014, after the reaction of different time gradient, is seen under microscope
It examines cellular morphology and photographs to record.
4. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
State the process following steps for being detected in SS01 and assessing the Skin Cell period in cell survival degree：
S021, human skin cutin strain cell culture is taken to be cultivated in 24 well culture plates at least 24 hours；
S022,10 μ L orchid growing point extracts are added, are reacted in incubator；
Supernatant and cell are taken out after S023, reaction, and are put into centrifuge tube and are centrifuged 5 minutes with 1200rpm；Supernatant is removed,
The PBS of 300 μ L is added, slowly shake and is moved in microcentrifugal tube after 700 μ L alcohol fixation cells are added dropwise, is placed in 4 DEG C
Refrigerator storage；
S024, microcentrifugal tube is centrifuged 5 minutes with 1200rpm, removes supernatant, the PBS of 445 μ L, 5 μ L concentration is sequentially added
For the ribonuclease A of 10mg/ml and the Triton X-100 of 50 μ L a concentration of 10%, reacted 30 minutes in 37 DEG C
The RNA of human skin cutin strain cell is destroyed after decomposing, after centrifuging 5 minutes with 1200rpm and remove supernatant, is added
The polyimide solution that the PBS of 400 μ L adds 5 a concentration of 5mg/ml of μ L after mixing is protected from light 5 at a temperature of 4 DEG C
Minute, to filter membrane filtration；
S025, using stream type cell analyzer and coordinate Winmdi softwares analyze cell cycle distribution ratio.
5. special according to a kind of any one big white orchid growing point extract safety detecting methods of claim 2-4
Sign is that cell density of the human skin cutin strain cell in 96 well culture plates is 1 × 104/well。
6. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
The process following steps of orchid growing point extract protection cyclic DNA effect in SS02 are estimated in commentary：
S0311, by pUC119DNA plasmids with 1:8 ratios are diluted with PBS；
S0312, the dilution in S0311 is taken to set up control group, ultraviolet light and hydrogen peroxide effect group, ultraviolet light and hydrogen peroxide respectively
Effect+extract group；
S0313, pUC119DNA plasmid dilutions of the 2 μ L through S0312 step process is respectively taken to be placed in microcentrifugal tube, observation is sentenced
Whether disconnected extract has protection skin histology uvioresistant and oxidative damage efficiency；
Wherein, the extract in the ultraviolet light and hydrogen peroxide effect+extract group is made into using orchid growing point extract
The extract liquor of various concentration gradient acts on 1 hour in 37 DEG C, molten in the agarose of 0.8% concentration after load dyestuff mixing is added
Electrophoresis is carried out in liquid, the percentage of S types DNA and L-type DNA are analyzed in latter electrophoresis film image acquisition system analysis in 30 minutes.
7. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
The process following steps of orchid growing point extract protected fragment DNA effects in SS02 are estimated in commentary：
S0321, the DNA fragmentation of diluted 2 μ L standard molecular weights is added in micro tube；
S0322, control group, ultraviolet light and hydrogen peroxide effect group, ultraviolet light and hydrogen peroxide effect+extract group are set up respectively；
S0323, the orchid growing point extract liquor for taking various concentration gradient act on 1 hour in 37 DEG C, and load dyestuff mixing is added
Afterwards, electrophoresis is carried out in the agarose solution of 0.8% concentration, S is analyzed in latter electrophoresis film image acquisition system analysis in 30 minutes
The percentage of type DNA and L-type DNA.
8. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
State the process following steps that extract maintenance Skin Cell cell survival degree after action of ultraviolet radiation is assessed in SS03：
S0411, take human skin cutin strain cell culture in 96 well culture plates, and 96 well culture plates be placed on 37 DEG C and
5%CO2It is cultivated in incubator at least 24 hours；
S0412, control group, ultraviolet light irradiation group, ultraviolet light irradiation+extract processing group are set up respectively；
S0413, after reaction removes culture solution, is cleaned with PBS and the MTT for rejoining new culture solution and 10ul is molten
Liquid reacts, at 37 DEG C and 5%CO2Culture reaction removes culture solution after 4 hours in incubator, and the DMSO dissolvings of 100 μ L are added
Jia Za sediments, finally measure light absorption value under wavelength 570nm, analyze the cell survival degree in each group.
9. a kind of big white orchid growing point extract safety detecting method according to claim 1, which is characterized in that institute
State the process following steps of the ring fourth pyrimidine dimer production quantity of assessment detecting DNA damage in SS03：
S0421, cell density is taken to be 1 × 105The human skin cutin strain cell culture of/well is cultivated in 24 well culture plates
At least 24 hours；
S0422, control group, ultraviolet light irradiation group, ultraviolet light irradiation+extract processing group are set up respectively；
S0423, treated cell is added in serum-free fresh medium and continues culture 4 hours, removed culture solution, use
PBS, which is cleaned and ice methanol is added, fixes cell, then is acted on Triton X-100, and the hydrochloric acid of a concentration of 2mol/l is added
Effect fills up space between cells with 1% bovine serum albumin(BSA) again after 1 hour, adds CPD Primary antibodies and is shaken under the conditions of 37 DEG C
Reaction 1 hour is shaken, CPD Primary antibodies is taken out, adds CPD secondary antibodies and be protected from light 30 minutes, cleaned with PBS, in wavelength
To detect fluorescence performance under conditions of 504-524nm；Add fluorescent dye carry out nuclear targeting, then to be cleaned with PBS after
Detecting fluorescence performance under conditions of wavelength is 355-460nm, and take pictures under the microscope in fluorescence microscopy；
S0424, detecting fluorescence performance under conditions of wavelength is 504-524nm to cell in each group, are 355-460nm in wavelength
Under conditions of detecting fluorescence performance and microscope under photo compare and analyze.
10. a kind of big white orchid growing point extract safety detecting method according to claim 9, which is characterized in that
The control group is to remove cell supernatant, replaces serum fresh medium, after cultivating 4 hours, is placed in nothing after being cleaned with PBS again
Continue culture 4 hours in serum fresh medium；
Wherein, the ultraviolet light irradiation group is to remove cell supernatant, replaces serum fresh medium, after cultivating 4 hours, is removed
It is cleaned and is drained with PBS after supernatant, after carrying out ultraviolet light irradiation respectively, be added in serum-free fresh medium continue to train immediately
It supports 4 hours；
Wherein, the ultraviolet light irradiation+extract processing group is to remove cell supernatant, replaces serum fresh medium, culture 4
It after hour, is cleaned and is drained with PBS after removing supernatant, after carrying out ultraviolet light irradiation respectively, the nothing containing extract is added immediately
Continue culture 4 hours in serum fresh medium.
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