CN1218837A - Detecting and screening method for biological active additive component of cosmetics - Google Patents
Detecting and screening method for biological active additive component of cosmetics Download PDFInfo
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- CN1218837A CN1218837A CN97119496A CN97119496A CN1218837A CN 1218837 A CN1218837 A CN 1218837A CN 97119496 A CN97119496 A CN 97119496A CN 97119496 A CN97119496 A CN 97119496A CN 1218837 A CN1218837 A CN 1218837A
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Abstract
In the said method, biological active additive component is detected and screened directly by extracorporeal healthy human and mouse epidermis cell culturing system and through observation of the physiological effect of the active component on epidermis cell. At the same time, the physilogical effect of the active component on epidermis cell is measured by means of porous cultivating plate for culturing epidermis cell and detecting and screening optimal concentration gradient, radioisotope incorporation label; in situ embedding of cultured cell, and the proliferaion, growth, senility and peeling off of the epidermis cell.
Description
The present invention relates to adopt biological method to detect and screening makeup biological activity effective ingredient
Raising along with living standards of the people, people increase day by day to the demand of makeup, makeup market develops rapidly, various makeup kinds and formulation flood market, meanwhile the skin adverse reaction that causes because of makeup has become a kind of public hazards, and it directly affects human consumer's physical and mental health.Therefore the evaluation of further strengthening cosmetics quality is very necessary.The present invention is directed to the problem of present existence, develop a kind of biological active additive component of cosmetics detection and screening method.
The object of the invention is, utilizes normal people and mouse skin cells in vitro culture systems, directly biological active additive component of cosmetics is detected and screens, and observes the physiological action of the biological active additive component of various makeup to epidermic cell.Adopted four kinds of evaluation meanses to weigh the physiological action of biological active additive component of cosmetics simultaneously to epidermic cell; (1) the porous culture plate is cultivated epidermic cell and is detected and screen biological active additive component of cosmetics optimum concn gradient; (2) radio isotope mixes mark and observes the influence of biological active additive component of cosmetics to epidermic cell dna replication dna and protein synthesis; (3) the epidermic cell embedding in situ of Pei Yanging, electron microscopic section are observed epidermic cell form and organoid structure; (4) the epidermal cell proliferation of cultivation, in growth, aging and the process that comes off, following the tracks of fixing dyeing optics microscopically observes, estimate the influence of biological active additive component of cosmetics to the epidermic cell growth cycle, by detection and the screening of adopting this method, can determine the value of makeup biological activity effective ingredient fully.
The detection of biological active additive component of cosmetics of the present invention and screening method, this method at first utilize normal people and mouse skin cells in vitro culture systems, directly makeup biological activity effective ingredient are detected and screen; In detection and screening process, adopt the porous culture plate to cultivate epidermic cell; Radio isotope mixes mark; The epidermic cell embedding in situ of cultivating, the section of electric border, at the epidermal cell proliferation of cultivating, growth is in the aging and the process that comes off, follow the tracks of evaluation meanses such as fixing dyeing optics microscopically observation, weigh the physiological action of biological active additive component of cosmetics epidermic cell; Normal people and mouse skin cells in vitro culture systems are to adopt following method: (a), with 199 substratum, the Eagle substratum, the RPNI-1640 substratum, the NEN substratum, the DNEN substratum, wherein a kind of is basic medium, use collagen gel, mouse 313 cells, human dermis inoblast and mix one of them trophoderm of fibroblastic collagen gel for epidermic cell cultivation, in nutrient solution, add 5-20% calf serum or foetal calf serum, add protein growth factor (Urogastron, Toxins,exo-, cholera, Regular Insulin etc.) and steroid hormone (hydrocortisone, 16-Methylprednisone acetate etc.) will digest, the epidermal basal cell suspension inoculation that filtration and centrifugal back obtain is in the nutrient solution of PH5-8, and controlled temperature 36-38 ℃ gets final product.(b), with selective medium (as the NCD8153 substratum) is basic medium, use collagen gel, mouse 313 cells, human dermis inoblast and mixed wherein a kind of trophoderm of fibroblastic collagen gel for epidermic cell cultivation, add the pituitary body extract, add protein growth factor, (Urogastron, Toxins,exo-, cholera, Regular Insulin etc.) and steroid hormone (hydrocortisone, 16-Methylprednisone acetate etc.), to digest, the epidermal basal cell suspension inoculation that filtration and centrifugal back obtain is in the nutrient solution of PH5-75, and controlled temperature 36-38 ℃ gets final product.
Adopt following four kinds of evaluation meanses then, weigh the physiological action of biological active additive component of cosmetics epidermic cell:
(1) normal people or mouse skin cell inoculation are cultivated on the porous culture plate, under same culture condition, in nutrient solution, add the biological active additive component of cosmetics sample that will detect and screen, and according to the hole count of porous culture plate, the concentration gradient that setting will detect and screen, according to the every hole of the porous culture plate mesocuticle cell epidermis diaphragm area that the dyeing back forms in limiting fate, thickness is judged the optimum concn gradient of wanting to have or not in the test sample or promote epidermal cell proliferation;
(2) pass through tritium-labeled nucleosides (as tritium-labeled thymidine H
3-Thynuidine) and tritium-labeled amino acid (tritium-labeled leucine H
3-Leu) in the epidermic cell culturing process, mix the biological active additive component of cosmetics that situation detects and screens, under various dose, promote dna replication dna and protein synthesis situation, weigh whether have trophism;
(3), the different time of cultivating at epidermic cell, get the epidermic cell of cultivating under the different biological active additive component of cosmetics concentration, carry out embedding in situ, electron microscopic section is observed, and understands the influence of biological active additive component of cosmetics to epidermic cell keratinization degree, organoid structure, the multiple stratification of epidermis diaphragm;
(4) with normal people or mouse skin cell cultures in culturing bottle, culture dish, place different makeup biological activity effective ingredient work to add under the agent concentration, every 1-2 days, cell fixation is dyeed, the biomorph that carries out observing epidermal cell proliferation, growth, aging under the opticmicroscope and come off changes, and can estimate the effect and the value of biological active additive component of cosmetics in sum by four kinds of means.
Embodiment 1:
(1) at first set up the external epidermic cell culture systems of mouse:
Getting 199 substratum is basic medium, with the trophoderm of collagen gel as the epidermic cell cultivation, in nutrient solution, add 5% calf serum, add Regular Insulin and hydrocortisone then, the epidermal basal cell suspension inoculation that digestion, filtration and centrifugal back are obtained is in the nutrient solution of PH5-8 again, controlled temperature 36-38 ℃, can build up stripped mouse skin cell culture system, directly biological active additive component of cosmetics is detected and screens, observe the physiological action of various active additive components epidermic cell.
(2) adopt following four kinds of evaluation meanses to weigh the physiological action of biological active additive component of cosmetics to epidermic cell.
(a), the porous culture plate is cultivated epidermic cell and is detected and screen biological active additive component of cosmetics optimum concn gradient.
(b), radio isotope mixes mark and observes biological active additive component of cosmetics to epidermic cell dna replication dna and protein synthesis influence.
(c), the epidermic cell embedding in situ of Pei Yanging, electron microscopic section are observed epidermic cell form and organoid structure.
(d), in epidermal cell proliferation, growth, aging of cultivating and the process that comes off, follow the tracks of fixing dyeing optics microscopically and observe, the evaluation biological active additive component of cosmetics is to the influence of epidermic cell growth cycle.
Embodiment 2:
(1) at first set up the external epidermic cell culture systems of mouse:
Getting the Eagle substratum is basic medium, with the trophoderm of mouse 313 cells as the epidermic cell cultivation, in nutrient solution, add 10% bovine serum, the low cell suspension inoculation of epidermis base that digestion, filtration and centrifugal back are obtained is in the nutrient solution of PH5-8 again, controlled temperature 36-38 ℃, can build up stripped mouse skin cell culture system, directly biological active additive component of cosmetics be detected and screens, observe the physiological action of various active additive components epidermic cell.
(2) adopt following four kinds of evaluation meanses to weigh the physiological action of biological active additive component of cosmetics to epidermis.
(a), the porous culture plate is cultivated epidermic cell and is detected and screen biological active additive component of cosmetics optimum concn gradient.
(b), radio isotope mixes mark and observes biological active additive component of cosmetics to epidermic cell dna replication dna and protein synthesis influence.
(c), the epidermic cell embedding in situ of Pei Yanging, electron microscopic section are observed epidermic cell form and organoid structure.
(d), in epidermal cell proliferation, growth, aging of cultivating and the process that comes off, follow the tracks of fixing dyeing optics microscopically and observe, the evaluation biological active additive component of cosmetics is to the influence of epidermic cell growth cycle.
Embodiment 3:
(1) at first set up normal human's exterior skin cell culture system:
Getting the DNEN substratum is basic medium, the trophoderm that the personnel selection dermal fibroblast is cultivated as epidermic cell, in nutrient solution, add 20% calf serum, add Urogastron, hydrocortisone then, the low cell suspension inoculation of epidermis base that digestion, filtration and centrifugal back are obtained is in the nutrient solution of PH5-8 again, controlled temperature 36-38 ℃, can build up stripped normal people's epidermic cell culture systems, directly biological active additive component of cosmetics is detected and screens, observe the physiological action of various active additive components epidermic cell.
(2) adopt following four kinds of evaluation meanses to weigh the physiological action of biological active additive component of cosmetics to epidermic cell.
(a) the porous culture plate is cultivated epidermic cell and is detected and screen biological active additive component of cosmetics optimum concn gradient.
(b), radio isotope mixes mark and observes biological active additive component of cosmetics to epidermic cell dna replication dna and protein synthesis influence.
(c), the epidermic cell embedding in situ of Pei Yanging, electron microscopic section are observed epidermic cell form and organoid structure.
(d), in epidermal cell proliferation, growth, aging of cultivating and the process that comes off, follow the tracks of fixing dyeing optics microscopically and observe, the evaluation biological active additive component of cosmetics is to the influence of epidermic cell growth cycle.
Embodiment 4:
(1) at first set up normal human's exterior skin cell culture system:
Getting the RPN1-1640 substratum is basic medium, with mixing the trophoderm that fibroblastic collagen gel is cultivated as epidermic cell, in nutrient solution, add 10% foetal calf serum, add Urogastron and hormone 16-Methylprednisone acetate then, to digest again, the low cell suspension inoculation of epidermis base that filtration and centrifugal back obtain is in the nutrient solution of PH5-8, controlled temperature 36-38 ℃, can build up stripped normal people's epidermic cell culture systems, directly biological active additive component of cosmetics is detected and screens, observe the physiological action of various active additive components epidermic cell.
(2) adopt following four kinds of evaluation meanses to weigh the physiological action of biological active additive component of cosmetics to epidermic cell.
(a) the porous culture plate is cultivated epidermic cell and is detected and screen biological active additive component of cosmetics optimum concn gradient.
(b), radio isotope mixes mark and observes biological active additive component of cosmetics to epidermic cell dna replication dna and protein synthesis influence.
(c), the epidermic cell embedding in situ of Pei Yanging, electron microscopic section are observed epidermic cell form and organoid structure.
(d), in epidermal cell proliferation, growth, aging of cultivating and the process that comes off, follow the tracks of fixing dyeing optics microscopically and observe, the evaluation biological active additive component of cosmetics is to the influence of epidermic cell growth cycle.
Embodiment 5:
(1) at first set up the external epidermic cell culture systems of normal people or mouse:
Getting the NCDB153 substratum is basic medium, with the trophoderm of collagen gel as the epidermic cell cultivation, add the pituitary body extract, add Toxins,exo-, cholera, hydrocortisone then, the low cell suspension inoculation of epidermis base that digestion, filtration and centrifugal back are obtained is in the nutrient solution of PH5-8 again, controlling moisture 36-38 ℃, can build up stripped normal people or mouse skin cell culture system, directly biological active additive component of cosmetics is detected and screens, observe the physiological action of various active additive components epidermic cell.
(2) adopt following four kinds of evaluation meanses to weigh the physiological action of biological active additive component of cosmetics to epidermic cell.
(a) the porous culture plate is cultivated epidermic cell and is detected and screen biological active additive component of cosmetics optimum concn gradient.
(b), radio isotope mixes mark and observes biological active additive component of cosmetics to epidermic cell dna replication dna and protein synthesis influence.
(c), the epidermic cell embedding in situ of Pei Yanging, electron microscopic section are observed epidermic cell form and organoid structure.
(d), in epidermal cell proliferation, growth, aging of cultivating and the process that comes off, follow the tracks of fixing dyeing optics microscopically and observe, the evaluation biological active additive component of cosmetics is to the influence of epidermic cell growth cycle.
Claims (3)
1, a kind of detection of biological active additive component of cosmetics and screening method is characterized in that, this method is utilized normal people or mouse skin cells in vitro culture systems, directly makeup biological activity effective ingredient is detected and screens; Adopt porous maintenance plate to cultivate epidermic cell, radio isotope mixes mark; Epidermic cell embedding in situ, the electric border sections observation of cultivating, at the epidermal cell proliferation of cultivating, growth is in the aging and the process that comes off, follow the tracks of evaluation meanses such as fixing dyeing observation by light microscope, weigh the physiological action of biological active additive component of cosmetics epidermic cell.
2, the detection of biological active additive component of cosmetics according to claim 1 and screening method is characterized in that, normal people or mouse skin cells in vitro culture systems are to adopt following method to build up:
(a), with 199 substratum, the Fagle substratum, the RPHI-1640 substratum, the WEN substratum, the ONEN substratum, wherein a kind of is basic medium, use collagen gel, mouse 313 cells, human dermis inoblast and mix one of them trophoderm of fibroblastic collagen gel for epidermic cell cultivation, in nutrient solution, add 5-20% calf serum or foetal calf serum, add protein growth factor (Urogastron, Toxins,exo-, cholera, Regular Insulin etc., and steroid hormone (hydrocortisone, 16-Methylprednisone acetate etc.), to digest, the epidermal basal cell suspension inoculation that filtration and centrifugal back obtain is in the nutrient solution of PH5-8, and controlled temperature 36-38 ℃ gets final product.
(b), with selective medium (as the NCDB153 substratum) is basic medium, use collagen gel, mouse 373 cells, human dermis inoblast and mixed wherein a kind of trophoderm of fibroblastic collagen gel for epidermic cell cultivation, add the pituitary body extract, add protein growth factor, (Urogastron, Toxins,exo-, cholera, Regular Insulin etc.) and hormone (hydrocortisone, 16-Methylprednisone acetate etc.), to digest, the epidermal basal cell suspension inoculation that filtration and centrifugal back obtain is in the nutrient solution of PH5-7.5, and controlled temperature 36-38 ℃ gets final product.
3, the detection of biological active additive component of cosmetics according to claim 1 and screening method is characterized in that, adopt following four kinds of evaluation meanses, weigh the physiological action of active additive component to epidermic cell;
(1) normal people or mouse skin cell inoculation are cultivated on the porous culture plate, under same culture condition, in nutrient solution, add the biological active additive component of cosmetics sample that will detect and screen, and according to the hole count of porous culture plate, the concentration gradient that setting will detect and screen, according to the every hole of the porous culture plate mesocuticle cell epidermis diaphragm area that the dyeing back forms in limiting fate, thickness is judged the optimum concn gradient of wanting to have or not in the test sample or promote epidermal cell proliferation;
(2) pass through tritium-labeled nucleosides (as tritium-labeled thymidine H
3-Ibynoidine) and tritium-labeled amino acid (tritium-labeled leucine H
3-Leu) in the epidermic cell culturing process, mix the biological active additive component of cosmetics that situation detects and screens, under various dose, promote dna replication dna and protein synthesis situation, weigh it and whether have trophism;
(3), the different time of cultivating at epidermic cell, get the epidermic cell of cultivating under the different biological active additive component of cosmetics concentration, carry out embedding in situ, electron microscopic section is observed, and understands the influence of biological active additive component of cosmetics to epidermic cell keratinization degree, organoid structure, the multiple stratification of epidermis diaphragm;
(4) with normal people or mouse skin cell cultures in culturing bottle, culture dish, place different makeup biological activity effective ingredient work to add under the agent concentration, every 1-2 days, with cell fixation dyeing, the biomorph variation of carrying out observing epidermal cell proliferation, growth, aging under the opticmicroscope and coming off.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97119496A CN1063226C (en) | 1997-12-02 | 1997-12-02 | Detecting and screening method for biological active additive component of cosmetics |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97119496A CN1063226C (en) | 1997-12-02 | 1997-12-02 | Detecting and screening method for biological active additive component of cosmetics |
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| CN1218837A true CN1218837A (en) | 1999-06-09 |
| CN1063226C CN1063226C (en) | 2001-03-14 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018192152A1 (en) * | 2017-04-19 | 2018-10-25 | 中国食品药品检定研究院 | Preparation and application of model for use in detecting in vitro endothelialization of vascular stent material |
| CN111337494A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Preparation method and application of artificial epidermis cosmetic detection kit |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1193168A1 (en) * | 1983-11-03 | 1985-11-23 | Институт биологической физики АН СССР | Method of obtaining man's epiderma cell culture |
| CN1039825C (en) * | 1990-10-13 | 1998-09-16 | 哈尔滨市卫生防疫站 | Glue membrane for measurement of total of colony |
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1997
- 1997-12-02 CN CN97119496A patent/CN1063226C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018192152A1 (en) * | 2017-04-19 | 2018-10-25 | 中国食品药品检定研究院 | Preparation and application of model for use in detecting in vitro endothelialization of vascular stent material |
| CN111337494A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Preparation method and application of artificial epidermis cosmetic detection kit |
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| CN1063226C (en) | 2001-03-14 |
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