CN108531543A - A kind of external combined method of evaluation hair tonic/Anti-hair loss effect - Google Patents
A kind of external combined method of evaluation hair tonic/Anti-hair loss effect Download PDFInfo
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- 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
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Abstract
The invention discloses a kind of external combined methods of evaluation hair tonic/anticreep effect, comprise the steps of：S1, the analysis of 5 alpha-reductase suppression levels；S2, cell efficiency analysis：It is analyzed containing cytotoxicity, ability of cell proliferation and oxidation resistance；S3, angiogenesis analysis；S4, evaluation of result and prediction.The present invention is used for the screening of both effectiveness cosmetic material by various biological means by a group synthase levels test, cell tests and tissue horizontal checkout；Simple system, cheap is tested, is not necessarily to special installation, detection method is quick, sensitive, general.The present invention can qualitative detection and evaluation hair tonic raw material the effect of, the method for the present invention can also replace living animal and human volunteer, be used for hair tonic and Anti-hair loss material toxicity and effect screening and detection.
Description
Technical field
The present invention relates to a kind of external combined methods of evaluation hair tonic/Anti-hair loss effect, can substitute living animal and be used for
Effect of new raw material, novel substance etc. is evaluated.
Background technology
Alopecia has become a kind of one of common phenomenon of puzzlement modern, and there are about 1.6 hundred million people depths in China according to the relevent statistics
Perplexed by alopecia.From the age, youth after 80s after 90s has become the group most perplexed by alopecia problem.It is reported that
Between 2013-2018, the estimated compound annual growth rate development by with 4% of global hair care industry, market value will be in the end of the year 2018
Reach 60,000,000,000 dollars.
Tradition educates hair evaluation method using zoopery, it is big with uncertainty, influence factor is more, the period is long,
The shortcomings of of high cost, and the requirement based on animal welfare, not only bad for realizing the quick, effective, high of raw material or novel substance
Flux screening, and need to use many experiments animal, run counter to the principle of reduction, optimization and replacement.In-vitro method is a kind of
Using in vitro tissue, organ etc. as experimental system, the method by being reacted in different means evaluations, predictor.With height
The advantages that flux, more terminals, high condition controllability, be conducive to the fast and effective screening of initial stage of development, but single method there is also
The deficiencies of predictive ability is limited.
II type, 5 alpha-reductase is present in the positions such as skin, prostate, can convert androgenic testosterone to protona,
To inhibit hair rest period to growth period transition, hair follicle normal growth is influenced, causes fat secretion excessive, male is caused to swash
Plain alopecia, 90% alopecia type is male sex hormone alopecia.Tested material inhibits the activity of 5 alpha-reductases that can inhibit double hydrogen in vitro
The generation of testosterone achievees the effect that reduce alopecia to inhibit protona to impact cell.
Dermal papilla cell (Dermal Papillary Cell, DPC) is the key cells of hair follicle growth, horn cell
It secretes cutin fiber and forms hair, the activity of cell and period are to evaluate whether the important indicator with cell Proliferation is promoted.Oxygen
It stress be considered as leading to cell ageing and dead one of the major reasons to change, the cell damage that antagonism/protection oxidative stress is brought
Wound has the significance for maintaining cell normal configuration and function.
Improving microcirculation can promote capilary to be formed, and restore blood supply, improve the nutrition of cell, and cell is promoted to increase
Grow the purpose with differentiation.The chorioallantoic membrane of chicken embryo, rich blood vessel, apparent, whether observable can promote capilary to give birth to
At, evaluate its improve microcirculation ability.
China's traditional plant is resourceful, greatly develops plant material in recent years, and many plant materials are declared to have apparent
The effect of act on, wherein just include hair tonic/Anti-hair loss effect.For more and more plant extracts, new synthetic,
Safety has received widespread attention with effect.
Invention content
The technical problems to be solved by the invention are just to provide a kind of external combination side of evaluation hair tonic/Anti-hair loss effect
Method contributes to exploitation to have effects that the product of clear hair tonic/Anti-hair loss, realizes quick, effective screening of the raw materials such as plant,
Save time and human cost.
Above-mentioned technical problem is solved, the technical solution adopted in the present invention is as follows.
A kind of external combined method of evaluation hair tonic/Anti-hair loss effect, it is characterized in that including the following steps：
S1, the analysis of 5 alpha-reductase suppression levels；S2, cellular level efficiency analysis：Including cytotoxicity, cell Proliferation energy
Power and oxidation resistance efficiency analysis；S3, angiogenesis analysis.
The step S1 includes following sub-step：
S1-1, preparation of reagents：
Stock：Phosphate buffer (pH=7.0), zyme extract, testosterone solution, NADPH solution, Finasteride solution and
Coomassie Brillant Blue solution；
Coomassie brilliant blue is prepared：Precision weighs Coomassie brilliant G-250 100mg, and 95% ethyl alcohol 50mL is added, and solution is in
Blue；Stirring and dissolving adds 85% (W/V) the phosphate buffer 100mL stirrings of pH=7.0, and solution is in blood red；Finally
It is settled to 1000mL with distilled water, solution becomes brown；It is stirred overnight, is filtered with filter paper spare on magnetic force heating stirrer；
Protein standard solution is prepared：It weighs bovine serum albumin(BSA) and is formulated as 0.1mg/mL protein standard liquids, be placed in 4
It is preserved in DEG C refrigerator；
Zyme extract contains：0.32mol/L sucrose, 1mmol/L Isosorbide-5-Nitraes-dithiothreitol dithio, 0.1mmol/L EDTA,
10mmol/L Tric-HCl and 20mmol/L phosphate buffers；
It is prepared by S1-2, II type, 5 alpha-reductase：
The male SD rat 4 for taking 250-300g, CO after being deprived of food but not water overnight2Humanity is put to death, and forefront is taken out in separation
Gland removes excess tissue, is shredded on Yu Bingtai；With the phosphate buffer of precooling 1 is made in glass homogenizer：5 homogenate,
10000g (parameter of centrifuge), 10min are centrifuged twice, take supernatant that phosphoric acid buffer is added after removing upper layer white adipose layer
Liquid is settled to 20mL, dispenses into EP pipes, often pipe 1mL, as thick enzyme extract；- 80 DEG C of refrigerators save backup；
S1-3, protein quantification：
Specification Curve of Increasing：0.1mg/mL BSA titers 0.1,0.2,0.4,0.6,0.8,1mL are taken, adds steaming respectively
Distilled water adds Coomassie brilliant G-250 solution 5mL to 1mL, mixing, after the 5min that develops the color 595nm at measurement light absorption value, paint
The standard curve of protein processed；It is reacted instead of BSA with thick enzyme, measures thick enzyme absorbance value, substituted into standard curve and obtain
Total protein content；
S1-4, the reaction of 5 alpha-reductases：
Contain 0.5mL phosphate buffers, 0.1mL50% tested materials or reference material in reaction system, 150 μ L testosterone solution,
The thick enzyme extracts of 250 μ L, 0.5mL and 2mg/mL NADPH solution；
30min is reacted at 37 DEG C, 2mL dichloromethane is added after reaction to be made reaction stop and extract testosterone, then so
The 100 μ g/mL propyl hydroxybenzoates of 0.25mL are added afterwards as internal standard, rocks 60s, is centrifuged under 400 × g centrifugal force
10min；Remove upper strata aqueous phase, remove 1mL organic phases simultaneously be evaporated, residue is dissolved in 1.5mL methanol, take 10 μ L efficient liquid phases into
Sample measures residual testosterone；
Enzyme reaction pipe, enzyme blank tube, standard quality control and sample cell are set simultaneously；Dichloromethane is added before reaction starts
So that enzyme is inactivated, is set as enzyme blank tube；Tested material is substituted with 50% ethyl alcohol of 0.1mL, is set as enzyme reaction pipe；
S1-5,5 alpha-reductase inhibiting rates measure:
Chromatographic condition：Chromatographic column：Luna C18 (2) column (4.6 × 250mm, 5 μm)；
Column temperature：40 DEG C, sample size：10 μ L, flow velocity：1mL/min, mobile phase：70% methanol
Detection wavelength：242nm；
S1-6, interpretation of result:
The testosterone concentration of enzyme reaction pipe is denoted as TReaction, the testosterone concentration of enzyme blank tube is denoted as TBlank, the testosterone concentration of sample cell
It is denoted as TSample；The ratio of testosterone and internal standard peak area is simultaneously denoted as r；
The step S2 includes following sub-step：
S2-1, cytotoxicity：
S2-1-1, preparation of reagents
Stock：10%FBS cell culture mediums, 0.25% pancreatin -0.02%EDTA, PBS and DMSO；
MTT solution is prepared：MTT 0.5g are weighed, the 0.01mol/L phosphate buffers for being dissolved in 100mL are configured to 5mg/mL
Solution, 0.22 μm of filter filtration sterilization, packing, 4 DEG C are kept in dark place；
It is prepared by S2-1-2, cell：
The cell that 80%-90% fusions will be grown to is digested with 0.25% pancreatin -0.02%EDTA, 1200rpm/min,
3-5min is centrifuged, and suspension, density 1.0 × 10 is made with complete medium in obtained cell precipitation4/ hole (per hole 100uL) inoculation
In 96 orifice plates；In 37 DEG C, 5%CO2It is cultivated 1-2 days in the incubator under saturated humidity, waits for that cell is grown to 80% and converge；
S2-1-3, exposure：
Tested material complete medium dissolved dilution to concentration to be measured；Culture medium original in 96 orifice plates is removed, per hole
The fresh culture mediums containing tested material of 200uL, at least eight series concentration is added, while setting up Positive control wells：There is cell,
Tested material is 5%SDS；Negative control hole：There is cell, is not added with tested material；Blank well：It is acellular, it is not added with tested material, culture solution；
24 and 48h is cultivated in incubator；
S2-1-4, cytoactive detection：
The MTT solution 20uL of 5mg/mL are added per hole, is incubated 4h in incubator, removes culture medium, DMSO is added in every hole
150uL is incubated 30min in incubator, measures the absorbance in often hole after mixing well 570nm at microplate reader；
S2-1-5, interpretation of result：
Inhibiting rate curve is drawn, the half-inhibition concentration (IC of tested material is calculated50) and 20% inhibition concentration (IC20)；
S2-2, ability of cell proliferation：
S2-2-1, preparation of reagents：
Cell culture medium containing 10%FBS, 0.25% pancreatin -0.02%EDTA, PBS, MTT solution, DMSO maintain culture medium
(cell culture medium containing 0-2%FBS).
It is prepared by S2-2-2, cell：
The cell that 80%-90% fusions will be grown to is digested with 0.25% pancreatin -0.02%EDTA, 1200rpm/min,
3-5min is centrifuged, and suspension, density 2.0-3.0 × 10 are made with complete medium in obtained cell precipitation4/ mL is (per hole
100uL) it is inoculated in 96 orifice plates or density 1 × 105/ mL is inoculated in 6 orifice plates (per hole 2mL)；At 37 DEG C, 5%CO2, saturated humidity
Under incubator in cultivate 18-24h；；It replaces and maintains culture solution, continue to cultivate 18-24h.
S2-2-3, exposure：
Old culture medium in hole is removed, the culture medium of the tested material containing various concentration is added, tested material maximum concentration is IC20, to
7 series concentrations of lower setting, and blank control group is set；Exposure duration 24 and 48h；
S2-2-4, ability of cell proliferation measure：
In 96 porocyte culture plates after exposure, the MTT solution 20uL of 5mg/mL are added per hole, 4 are incubated in incubator
Hour, culture medium is removed, DMSO 150uL are added in every hole, is incubated 30min in incubator, is existed with microplate reader after mixing well
The absorbance per hole is measured at 570nm；
S2-2-5, interpretation of result：
Cell viability
Each group carries out statistical analysis (α=0.05) with control group；
S2-3, oxidation resistance：
S2-3-1, preparation of reagents：
Cell culture medium containing 10%FBS, 0.25% pancreatin -0.02%EDTA, PBS, hydrogen peroxide (H2O2), MTT solution,
DMSO；
It is prepared by S2-3-2, cell：
The cell that 80%-90% fusions will be grown to is digested with 0.25% pancreatin -0.02%EDTA, 1200rpm/min,
3-5min is centrifuged, and suspension, density 1 × 10 is made with complete medium in obtained cell precipitation4/ mL (per hole 100uL) inoculation
In 96 orifice plates or density 1 × 105/ mL is inoculated in 6 orifice plates (per hole 2mL)；At 37 DEG C, 5%CO2, the incubator under saturated humidity
Middle culture 18-24h；
S2-3-3, exposure：
The former culture medium of removal, is added the culture medium of the tested material containing various concentration, hydrogen peroxide need to be added and cause cellular oxidation
Damage, while control group is set；Exposure duration is 24 and 48h；
S2-3-4, cytoactive detection：
The MTT solution 20uL of 5mg/mL are added per hole, is incubated 4 hours in incubator, removes culture medium, are added in every hole
DMSO 150uL are incubated 30min in incubator, measure the absorbance in often hole after mixing well 570nm at microplate reader；
S2-3-5, determination oxidative：
Using dichlorofluorescein (DCFH-DA) labelling method；DCFH-DA is a kind of fluorescence probe, can specificity with
ROS is combined, and when intracellular ROS level increases, the fluorescence intensity of DCFH-DA increases accordingly, is shown as relative to normal thin
The raising of born of the same parents' fluorescence intensity；
It is inoculated in the cell of 6 orifice plates, cell is suspended with 0.25% pancreatin -0.02%EDTA digestion, and centrifugation is clear with cold PBS
It washes 2 times, adjustment cell density is 1 × 106/ mL uses flow cytomery with ROS kits；
S2-3-6, interpretation of result：
Cell viability
Cell viability curve is drawn, analysis tested material is to H2O2Cause the protective effect of oxidative damage；
ROS suppression levels
Mean fluorescence intensity (MFI)=fluorescence intensity geometric mean；
Relative intensity of fluorescence
S3, angiogenesis analyze S3-1, material and reagent：
SPF grades of fertilization chicken embryos, methanol, acetone, 10% neutral formalin fixer, VEGF immunohistochemical kits
It is prepared by S3-2, CAM：
Fertilization chicken embryo, which is placed in incubator, is incubated (37.5 DEG C, RH40-70%), is overturn daily, is checked chick embryo development
Situation；Take well-developed chicken embryo in the 7th day, alcohol disinfecting eggshell opens the region of an about 2cm × 2cm at gas chamber end；Carefully
Separated gas chamber film, is completely exposed CAM；
S3-3, exposure：
Diameter about 0.5cm qualitative filter paper pieces are taken, the less position of content-addressable memory face blood vessel is gently placed in, 50 μ L tested materials are added,
Hyaline membrane seals, and returns to incubator and is incubated 72h；
S3-4, vessels analysis：
Blood-vessel image is analyzed：Blood vessel is directly observed under stereoscope, or fixer (methanol is added from window：Acetone=1:
1), after fixed 20min, one layer of CAM is taken out with tweezers, is paved on slide, image is acquired, counts vessel branch point quantity；
Immunohistochemistry：After taking out CAM, 10% neutral formalin fixer is added, it is conventional to make 5 μm after fixing for 24 hours
The paraffin section of thickness, is operated, last mounting according to VEGF immunohistochemical kit specifications, and micro- sem observation is gone forward side by side
Row photodensitometry；
S3-5, interpretation of result：
Test group mean vascular branch point quantity is compared with control group, and carries out statistical analysis, α=0.05；
The test group VEGF OD values that are averaged are compared with control group, and carry out statistical analysis, α=0.05.
In the step S1, positive control is 5 alpha-reductase reductase inhibitors, Finasteride (CAS： 98319-
26-7) a concentration of 0.1-1.0mg/mL.
In the step S2, used cell includes that people source dermal papilla cell (hDPC), immortal human cutin are thin
Born of the same parents (HaCaT) and people's primary keratinocyte, used positive control is minoxidil, and concentration range is 0.001-1 μ
mol/L；
In the step S2, cytotoxicity and analysis of cell proliferation, used method include mtt assay, dimethyl diaminophenazine chloride
Method, CCK methods and fluorescein breakthrough method；
In the step S2, cellular anti-oxidant capacity analysis carries out oxidative damage modeling, concentration using hydrogen peroxide
Ranging from 5-40 μm of ol/L, modeling time are 6-18h；
Oxidative damage modeling is carried out using hydrogen peroxide in the step S2, is divided to two kinds of models：Respectively before modeling
Tested material is added after tested material and modeling is added, evaluates the guarantor of tested material cell oxidative damage caused by hydrogen peroxide respectively
Shield acts on and repair.
In the step S3, image analysis carries out Image Acquisition using body formula mirror, is divided with image analysis software
Analysis counts thin vessels branch point in filter paper periphery 2-3cm.
In the present invention, under determining experiment condition, tested material can inhibit 5 alpha-reductases active, promote cell Proliferation,
There is antagonism/protective effect to oxidative damage, microcirculation can be promoted, can be used as has effects that potential hair tonic/Anti-hair loss
Strong evidence, tested material can be used as the constituent for educating hair system.
Description of the drawings
Cytotoxicity schematic diagram of Fig. 1 various concentrations tested material to HaCaT；
Cell Proliferation schematic diagram (* of Fig. 2 various concentrations tested material to HaCaT：p<0.05)；
Fig. 3 .1 are 0h time points blood-vessel image (blank control)；
Fig. 3 .2 are time point blood-vessel image (10% sample) for 24 hours；
Fig. 3 .3 are 48h time points blood-vessel image (30% sample)；
Fig. 3 .4 are 72h time point blood-vessel images
(upper left 0h, upper right are lower-left 48h, bottom right 72h for 24 hours).
Specific implementation mode
The enzyme inhibition of 1 Ginger P.E of example
S1,5 alpha-reductases inhibit external model
S1-1, preparation of reagents：Phosphate buffer (pH=7.0), zyme extract, testosterone solution, NADPH solution, that non-hero
Amine aqueous solution and Coomassie Brillant Blue solution；
Coomassie brilliant blue is prepared：Precision weighs Coomassie brilliant G-250 100mg, and 95% ethyl alcohol 50mL is added, and solution is in
Blue；Stirring and dissolving adds 85% (W/V) the phosphate buffer 100mL stirrings of pH=7.0, and solution is in blood red；Finally
It is settled to 1000mL with distilled water, solution becomes brown；It is stirred overnight, is filtered with filter paper spare on magnetic force heating stirrer；
Protein standard solution is prepared：It weighs bovine serum albumin(BSA) and is formulated as 0.1mg/mL protein standard liquids, be placed in 4
It is preserved in DEG C refrigerator；
Zyme extract contains：0.32mol/L sucrose, 1mmol/L Isosorbide-5-Nitraes-dithiothreitol dithio, 0.1mmol/L EDTA,
10mmol/L Tric-HCl and 20mmol/L phosphate buffers；
Ginger juice extract：100g gingers are taken, are cleaned up, disinfection, broken instrument is squeezed the juice, and is collected ginger juice, was centrifuged
Filter takes supernatant, crosses film, and 1,3 butanediols and Phenoxyethanol is added, and is uniformly mixed to get ginger juice extract.Nothing when test
Bacterium water is diluted to 30%, 10% and 1% 3 concentration by volume fraction.
It is prepared by S1-2, II type, 5 alpha-reductase：
The male SD rat 4 for taking 250-300g, CO after being deprived of food but not water overnight2Humanity is put to death, and forefront is taken out in separation
Gland removes excess tissue, is shredded on Yu Bingtai；With the phosphate buffer of precooling 1 is made in glass homogenizer：5 homogenate,
10000g, 10min are centrifuged twice, take supernatant addition phosphate buffer to be settled to 20mL after removing upper layer white adipose layer, point
It is filled in EP pipes, often pipe 1mL, as thick enzyme extract；- 80 DEG C of refrigerators save backup；
S1-3, protein quantification：
Specification Curve of Increasing：0.1mg/mL BSA titers 0.1,0.2,0.4,0.6,0.8,1mL are taken, adds steaming respectively
Distilled water adds Coomassie brilliant G-250 solution 5mL to 1mL, mixing, after the 5min that develops the color 595nm at measurement light absorption value, paint
The standard curve of protein processed；It is reacted instead of BSA with thick enzyme, measures thick enzyme absorbance value, substituted into standard curve and obtain
Total protein content；
S1-4, the reaction of 5 alpha-reductases：
Contain 0.5mL phosphate buffers, 0.1mL50% tested materials or reference material in reaction system, 150 μ L testosterone solution,
The thick enzyme extracts of 250 μ L, 0.5mL and 2mg/mL NADPH solution；
30min is reacted at 37 DEG C, 2mL dichloromethane is added after reaction to be made reaction stop and extract testosterone, then so
The 100 μ g/mL propyl hydroxybenzoates of 0.25mL are added afterwards as internal standard, rocks 60s, is centrifuged under 400 × g centrifugal force
10min；Remove upper strata aqueous phase, remove 1mL organic phases simultaneously be evaporated, residue is dissolved in 1.5mL methanol, take 10 μ L efficient liquid phases into
Sample measures residual testosterone；
Enzyme reaction pipe, enzyme blank tube, standard quality control and sample cell are set simultaneously；Dichloromethane is added before reaction starts
So that enzyme is inactivated, is set as enzyme blank tube；Tested material is substituted with 50% ethyl alcohol of 0.1mL, is set as enzyme reaction pipe；
S1-5,5 alpha-reductase inhibiting rates measure:
Chromatographic condition：Chromatographic column：Luna C18 (2) column (4.6 × 250mm, 5 μm)；
Column temperature：40 DEG C, sample size：10 μ L, flow velocity：1mL/min, mobile phase：70% methanol
Detection wavelength：242nm；
S1-6, interpretation of result:
As a result it shows：10% and 1% 5 alpha-reductase of ginger juice extract pair has certain inhibiting effect, shows that it may
Have effects that inhibit male sex hormone alopecia.
2 angelica extract cell efficiency analysis of example
S2, cell efficiency analysis
S2-1, cytotoxicity
S2-1-1, preparation of reagents：10%FBS cell culture mediums, 0.25% pancreatin -0.02%EDTA, PBS and DMSO；
MTT solution is prepared：MTT 0.5g are weighed, the 0.01mol/L phosphate buffers for being dissolved in 100mL are configured to the solution of 5mg/mL,
0.22 μm of filter filtration sterilization, packing, 4 DEG C are kept in dark place；
S2-1-2 angelica extracts：It takes the Radix Angelicae Sinensis medicinal material of 100g, boiling water to clean 2-3 times, one is added according to certain solid-liquid ratio
Determine the ethyl alcohol of concentration, heating and refluxing extraction, filtrate is collected in filtering.It is concentrated under reduced pressure into certain crude drug concentration, 1,3 butanediols are added
And Phenoxyethanol, it is uniformly mixed, centrifuged film to get angelica extract.When cytotoxicity test in 0.5~80% range
8 concentration are set.When efficacy test, 5 concentration of selection are analyzed in non-toxic range.
It is prepared by S2-1-3, cell：
HaCaT cells 0.25% pancreatin -0.02%EDTA digestion, the 1200rpm/ of 80%-90% fusions will be grown to
Min, 3-5min are centrifuged, and suspension, density 1.0 × 10 is made with complete medium in obtained cell precipitation4/ hole is (per hole
100uL) it is inoculated in 96 orifice plates；At 37 DEG C, 5%CO2, cultivate 1-2 days in the incubator under saturated humidity, wait for cell grow to
80% converges；
S2-1-4, exposure：
Tested material complete medium dissolved dilution to concentration to be measured；Culture medium original in 96 orifice plates is removed, per hole
The fresh culture mediums containing tested material of 200uL, at least eight series concentration is added, while setting up Positive control wells：There is cell,
Tested material is 5%SDS；Negative control hole：There is cell, is not added with tested material；Blank well：It is acellular, it is not added with tested material, culture solution；
24 and 48h is cultivated in incubator；
S2-1-5, cytoactive detection：
The MTT solution 20uL of 5mg/mL are added per hole, is incubated 4h in incubator, removes culture medium, DMSO is added in every hole
150uL is incubated 30min in incubator, measures the absorbance in often hole after mixing well 570nm at microplate reader；
S2-2, ability of cell proliferation：
S2-2-1, preparation of reagents：DMEM culture mediums, 0.25% pancreatin -0.02%EDTA, PBS, MTT containing 10%FBS
Solution, DMSO maintain culture medium：DMEM culture mediums containing 1%FBS
It is prepared by S2-2-2, cell：
The cell that 80%-90% fusions will be grown to is digested with 0.25% pancreatin -0.02%EDTA, 1200rpm/min,
3-5min is centrifuged, and suspension, density 2.0-3.0 × 10 are made with complete medium in obtained cell precipitation4/ mL is (per hole
100uL) it is inoculated in 96 orifice plates or density 1 × 105/ mL is inoculated in 6 orifice plates (per hole 2mL)；At 37 DEG C, 5%CO2, saturated humidity
Under incubator in cultivate 18-24h；It replaces and maintains culture solution, continue to cultivate 18-24h.
S2-2-3, exposure：
Old culture medium in hole is removed, the culture medium of the tested material containing various concentration is added, tested material maximum concentration is IC20, to
7 series concentrations of lower setting, and blank control group is set；Exposure duration 24 and 48h；
S2-2-4, ability of cell proliferation measure：
In 96 porocyte culture plates after exposure, the MTT solution 20uL of 5mg/mL are added per hole, 4 are incubated in incubator
Hour, culture medium is removed, DMSO 150uL are added in every hole, is incubated 30min in incubator, is existed with microplate reader after mixing well
The absorbance per hole is measured at 570nm；
S2-2-5, interpretation of result：
Cytotoxicity result is shown in Fig. 1.
Cell proliferation results are shown in Fig. 2.
As a result it shows：Cell toxicity test shows that concentration exists<When 20%, apparent cell toxicant is not generated to horn cell
Property.Cell proliferation test shows that concentration generates obvious facilitation at 20%, to horn cell, relatively low with concentration,
Facilitation also weakens therewith.
3 tuber of multiflower knotweed extract of example promotes to improve blood circulation
S3, angiogenesis analysis
S3-1, sample preparation
Tuber of multiflower knotweed extract：It takes the tuber of multiflower knotweed medicinal material of 100g, boiling water to clean 2-3 times, a certain concentration is added according to certain solid-liquid ratio
Ethyl alcohol, heating and refluxing extraction, filtering, collect filtrate.It is concentrated under reduced pressure into certain crude drug concentration, 1,3 butanediols and benzene oxygen is added
Ethyl alcohol is uniformly mixed, centrifuged film to get tuber of multiflower knotweed extract.
It is prepared by S3-2, CAM
SPF grades of fertilization chicken embryos are placed in incubator and are incubated (37.5 DEG C, RH40-70%), overturn daily, check chicken
Embryonic development situation；Take well-developed chicken embryo in the 7th day, alcohol disinfecting eggshell opens the area of an about 2cm × 2cm at gas chamber end
Domain；Careful separation cameral mantle, is completely exposed CAM；
S3-3, exposure：
Diameter about 0.5cm qualitative filter paper pieces are taken, the less position of content-addressable memory face blood vessel is gently placed in, 50 μ L tested materials are added,
Concentration 10%, hyaline membrane sealing return to incubator and are incubated 72h；
S3-4, vessels analysis：
Blood vessel is directly observed under stereoscope, is acquired image, is done qualitative analysis.
S3-5, as a result：See attached drawing 3.
As a result it shows：Fig. 3 .1 show that negative control is without effect is obviously promoted, and Fig. 3 .2 and 3.3 are it can be seen that various concentration
Tested material has certain facilitation to angiogenesis, and facilitation is more apparent under high concentration.
Claims (10)
1. a kind of external combined method of evaluation hair tonic/Anti-hair loss effect, it is characterized in that 3 kinds of in-vitro methods are contained, it is specific to wrap
Include following steps：
S1, the analysis of 5 alpha-reductase suppression levels；S2, cellular level efficiency analysis：Including cytotoxicity, ability of cell proliferation and resist
Oxidability efficiency analysis；S3, angiogenesis analysis.
2. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 1, it is characterized in that：The step
Rapid S1 includes following sub-step：
S1-1, preparation of reagents：
Stock：PH=7.0 phosphate buffers, zyme extract, testosterone solution, NADPH solution, Finasteride solution and coomassie are bright
Blue solution；
Coomassie brilliant blue is prepared：Precision weighs Coomassie brilliant G-250 100mg, 95% ethyl alcohol 50mL is added, solution is in blue；
Stirring and dissolving adds 85% (W/V) the phosphate buffer 100mL stirrings of pH=7.0, and solution is in blood red；Finally with distillation
Water is settled to 1000mL, and solution becomes brown；It is stirred overnight, is filtered with filter paper spare on magnetic force heating stirrer；
Protein standard solution is prepared：It weighs bovine serum albumin(BSA) and is formulated as 0.1mg/mL protein standard liquids, be placed in 4 DEG C of refrigerators
Middle preservation；
Zyme extract contains：0.32mol/L sucrose, 1mmol/L Isosorbide-5-Nitraes-dithiothreitol dithio, 0.1mmol/L EDTA,
10mmol/L Tric-HCl and 20mmol/L phosphate buffers；
It is prepared by S1-2, II type, 5 alpha-reductase：
The male SD rat 4-6 for taking 250-300g only, is deprived of food but not water rear CO overnight2Humanity is put to death, and prostate is taken out in separation, goes
Except excess tissue, shredded on Yu Bingtai；With the phosphate buffer of precooling 1 is made in glass homogenizer：5 homogenate, centrifuge parameters
10000g, 10min are centrifuged twice, take supernatant addition phosphate buffer to be settled to 20mL after removing upper layer white adipose layer, point
It is filled in EP pipes, often pipe 1mL, as thick enzyme extract；- 80 DEG C of refrigerators save backup；
S1-3, protein quantification：
Specification Curve of Increasing：0.1mg/mL BSA titers 0.1,0.2,0.4,0.6,0.8,1mL are taken, adds distilled water respectively extremely
1mL, adds Coomassie brilliant G-250 solution 5mL, mixing, measures light absorption value, drafting albumen after the 5min that develops the color 595nm at
The standard curve of matter；It is reacted instead of BSA with thick enzyme, measures thick enzyme absorbance value, substituted into standard curve and obtain total protein
Content；
S1-4, the reaction of 5 alpha-reductases：
Contain 0.5mL phosphate buffers, 0.1mL50% tested materials or reference material, 150 μ L testosterone solution, 250 μ in reaction system
The thick enzyme extract of L, 0.5mL and 2mg/mL NADPH solution；
30min is reacted at 37 DEG C, 2mL dichloromethane is added after reaction to be made reaction stop and extract testosterone, is subsequently added
Enter the 100 μ g/mL propyl hydroxybenzoates of 0.25mL as internal standard, rocks 60s, 10min is centrifuged under 400 × g centrifugal force；Removal
Upper strata aqueous phase removes 1mL organic phases and is evaporated, and residue is dissolved in 1.5mL methanol, and 10 μ L efficient liquid phase sample introductions is taken to measure residual testis
Ketone；
Enzyme reaction pipe, enzyme blank tube, standard quality control and sample cell are set simultaneously；Dichloromethane, which is added, before reaction starts makes enzyme lose
It is living, it is set as enzyme blank tube；Tested material is substituted with 50% ethyl alcohol of 0.1mL, is set as enzyme reaction pipe；
S1-5,5 alpha-reductase inhibiting rates measure：
Chromatographic condition：Chromatographic column：Luna C18 (2) column (4.6 × 250mm, 5 μm)；
Column temperature：40 DEG C, sample size：10 μ L, flow velocity：1mL/min, mobile phase：70% methanol Detection wavelength：242nm；
S1-6, interpretation of result:
The testosterone concentration of enzyme reaction pipe is denoted as TReaction, the testosterone concentration of enzyme blank tube is denoted as TBlank, the testosterone concentration of sample cell is denoted as
TSample；The ratio of testosterone and internal standard peak area is simultaneously denoted as r；
3. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 1, it is characterized in that：
The step S2 includes following sub-step：
S2-1, cytotoxicity：
S2-1-1, preparation of reagents
Stock：10%FBS cell culture mediums, 0.25% pancreatin -0.02%EDTA, PBS and DMSO；
MTT solution is prepared：MTT 0.5g are weighed, the 0.01mol/L phosphate buffers for being dissolved in 100mL are configured to the molten of 5mg/mL
Liquid, 0.22 μm of filter filtration sterilization, packing, 4 DEG C are kept in dark place；
It is prepared by S2-1-2, cell：
Cell 0.25% pancreatin -0.02%EDTA digestion, 1200rpm/min, the 3-5min of 80%-90% fusions will be grown to
Suspension, density 1.0 × 10 is made with complete medium in centrifugation, obtained cell precipitation4/ hole is inoculated in 96 holes (per hole 100uL)
Plate；In 37 DEG C, 5%CO2It is cultivated 1-2 days in the incubator under saturated humidity, waits for that cell is grown to 80% and converge；
S2-1-3, exposure：
Tested material complete medium dissolved dilution to concentration to be measured；Culture medium original in 96 orifice plates is removed, is added per hole
The fresh culture mediums containing tested material of 200uL are arranged 6-8 series concentration, while setting up Positive control wells：There is cell, by
Examination object is 5%SDS；Negative control hole：There is cell, is not added with tested material；Blank well：It is acellular, it is not added with tested material, culture solution；Training
It supports and cultivates 24 and 48h in case；
S2-1-4, cytoactive detection：
The MTT solution 20uL of 5mg/mL are added per hole, is incubated 4h in incubator, removes culture medium, DMSO is added in every hole
150uL is incubated 30min in incubator, measures the absorbance in often hole after mixing well 570nm at microplate reader；
S2-1-5, interpretation of result：
Inhibiting rate curve is drawn, the half-inhibition concentration (IC of tested material is calculated50) and 20% inhibition concentration (IC20)；
S2-2, ability of cell proliferation：
S2-2-1, preparation of reagents：
Cell culture medium containing 10%FBS, 0.25% pancreatin -0.02%EDTA, PBS, MTT solution, DMSO maintain culture medium；
It is prepared by S2-2-2, cell：
Cell 0.25% pancreatin -0.02%EDTA digestion, 1200rpm/min, the 3-5min of 80%-90% fusions will be grown to
Suspension, density 2.0-3.0 × 10 are made with complete medium in centrifugation, obtained cell precipitation4/ mL is inoculated in every hole 100uL's
96 orifice plates or density 1 × 105/ mL is inoculated in the 6 orifice plates of every hole 2mL；At 37 DEG C, 5%CO2, in the incubator under saturated humidity
Cultivate 18-24h；；It replaces and maintains culture solution, continue to cultivate 18-24h；
S2-2-3, exposure：
Old culture medium in hole is removed, the culture medium of the tested material containing various concentration is added, tested material maximum concentration is IC20, to dividing into
6-8 series concentration is set, and blank control group is set；Exposure duration 24 and 48h；
S2-2-4, ability of cell proliferation measure：
In 96 porocyte culture plates after exposure, the MTT solution 20uL of 5mg/mL are added per hole, are incubated 4 hours in incubator,
Culture medium is removed, DMSO 150uL are added in every hole, 30min is incubated in incubator, after mixing well with microplate reader at 570nm
Measure the absorbance per hole；
S2-2-5, interpretation of result：
Cell viability
Each group carries out statistical analysis with control group：α=0.05；
S2-3, oxidation resistance：
S2-3-1, preparation of reagents：
Cell culture medium containing 10%FBS, 0.25% pancreatin -0.02%EDTA, PBS, hydrogen peroxide, MTT solution, DMSO；
It is prepared by S2-3-2, cell：
Cell 0.25% pancreatin -0.02%EDTA digestion, 1200rpm/min, the 3-5min of 80%-90% fusions will be grown to
Suspension, density 1 × 10 is made with complete medium in centrifugation, obtained cell precipitation4/ mL is inoculated in 96 holes of every hole 100uL
Plate or density 1 × 105/ mL is inoculated in the 6 orifice plates of every hole 2mL；At 37 DEG C, 5%CO2, cultivate in the incubator under saturated humidity
18-24h；
S2-3-3, exposure：
The former culture medium of removal, is added the culture medium of the tested material containing various concentration, hydrogen peroxide need to be added and cause cell oxidative damage,
Control group is set simultaneously；Exposure duration is 24 and 48h；
S2-3-4, cytoactive detection：
The MTT solution 20uL of 5mg/mL are added per hole, is incubated 4 hours in incubator, removes culture medium, DMSO is added in every hole
150uL is incubated 30min in incubator, measures the absorbance in often hole after mixing well 570nm at microplate reader；
S2-3-5, determination oxidative：
Using dichlorofluorescein (DCFH-DA) labelling method；DCFH-DA is a kind of fluorescence probe, can specificity with ROS tie
It closes, when intracellular ROS level increases, the fluorescence intensity of DCFH-DA increases accordingly, shows as relative to normal cell fluorescence
The raising of intensity；
It is inoculated in the cell of 6 orifice plates, cell is suspended with 0.25% pancreatin -0.02%EDTA digestion, and centrifugation cleans 2 with cold PBS
Secondary, adjustment cell density is 1 × 106/ mL uses flow cytomery with ROS kits；
S2-3-6, interpretation of result：
Cell viability
Cell viability curve is drawn, analysis tested material is to H2O2Cause the protective effect of oxidative damage；
ROS suppression levels
Mean fluorescence intensity (MFI)=fluorescence intensity geometric mean；
4. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 1, it is characterized in that：The step
Rapid S3 includes following sub-step：
S3-1, material and reagent：
Specific pathogen free (SPF grades) fertilization chicken embryos, methanol, acetone, 10% neutral formalin fixer, vascular endothelial growth factors
Sub (VEGF) immunohistochemical kit
It is prepared by S3-2, chorioallantoic membrane (CAM)：
Fertilization chicken embryo, which is placed in incubator, to be incubated, and 37.5 DEG C, RH40-70%, is overturn daily, is checked chick embryo development situation；
Take well-developed chicken embryo in the 7th day, alcohol disinfecting eggshell opens the region of an about 2cm × 2cm at gas chamber end；Careful separation gas
Room film, is completely exposed CAM；
S3-3, exposure：
Diameter about 0.5cm qualitative filter paper pieces are taken, the less position of content-addressable memory face blood vessel is gently placed in, 50 μ L tested materials, hyaline membrane is added
Sealing returns to incubator and is incubated 72h；
S3-4, vessels analysis：
Blood-vessel image is analyzed：Blood vessel is directly observed under stereoscope, or fixer is added from window：Methanol：Acetone=1:1, Gu
After determining 20min, one layer of CAM is taken out with tweezers, is paved on slide, image is acquired, counts vessel branch point quantity；
Immunohistochemistry：After taking out CAM, 10% neutral formalin fixer is added, after fixing for 24 hours, 5 μ m thicks of conventional making
Paraffin section is operated, last mounting, micro- sem observation according to VEGF immunohistochemical kit specifications, and it is close to carry out light
Degree analysis；
S3-5, interpretation of result：
Test group mean vascular branch point quantity is compared with control group, and carries out statistical analysis, α=0.05；
The test group VEGF OD values that are averaged are compared with control group, and carry out statistical analysis, α=0.05.
5. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 2, it is characterized in that：The step
In rapid S1, positive control is 5 alpha-reductase reductase inhibitors, Finasteride (CAS：98319-26-7) a concentration of 0.1-
1.0mg/mL。
6. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 3, it is characterized in that：The step
In rapid S2, used cell includes that people source dermal papilla cell (hDPC), immortal human horn cell (HaCaT) and people are primary
Horn cell.
7. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 3, it is characterized in that：The step
In rapid S2, used positive control is minoxidil, and concentration range is 0.001-1 μm of ol/L；Cytotoxicity and cell increase
Analysis is grown, used method includes mtt assay, dimethyl diaminophenazine chloride method, CCK methods and fluorescein breakthrough method.
8. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 3, it is characterized in that：The step
In rapid S2, cellular anti-oxidant capacity analysis carries out oxidative damage modeling using hydrogen peroxide, and concentration range is 5-40 μm of ol/L,
The modeling time is 6-18h.
9. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 3, it is characterized in that：The step
In rapid S2, oxidative damage modeling is carried out using hydrogen peroxide, is divided into two kinds of models：Tested material and modeling are added respectively before modeling
After tested material is added, respectively evaluate tested material cell oxidative damage caused by hydrogen peroxide protective effect and repair.
10. the external combined method of evaluation hair tonic/Anti-hair loss effect according to claim 4, it is characterized in that：The step
In rapid S3, the positive control used is the positive control (bFGF) of middle use, a concentration of 1000-3000U/mL；Image point
Analysis carries out Image Acquisition using body formula mirror, is analyzed with image analysis software, and thin vessels branch in filter paper periphery 2-3cm is counted
Point.
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