CN1063226C - 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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- CN1063226C CN1063226C CN97119496A CN97119496A CN1063226C CN 1063226 C CN1063226 C CN 1063226C CN 97119496 A CN97119496 A CN 97119496A CN 97119496 A CN97119496 A CN 97119496A CN 1063226 C CN1063226 C CN 1063226C
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Abstract
The present invention relates to a detecting and screening method for biological active additive effective components of cosmetics by a biochemical means. The method directly detects and screens the biological active additive effective components of cosmetics by making use of a normal person or an in vitro culture system for mouse epidermal cells, a person observes physiological action of the additive effective components for epidermal cells, and simultaneously, a porous culture board is adopted to culture and detect epidermal cells and to screen the optimal concentration gradient; radioisotope is incorporated in label; cultured epidermal cells are embedded at the original position; a method of epidermal cell proliferation, growth, ageing and shedding is adopted, an evaluation means measures the physiological action of the additive effective components for the epidermal cells.
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 to epidermic cell simultaneously: (1) 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 RPN1-1640 substratum, the NFN substratum, the DNFN substratum, wherein a kind of is basic medium, use collagen gel, mouse 3T3 cell, 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 3T3 cell, 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 PH5-7.5, 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) in the epidermic cell culturing process, mix the biological active additive component of cosmetics that situation detects and screens by tritium-labeled nucleosides (as tritium-labeled thymidine H3-Thynoidine) and tritium-labeled amino acid (tritium-labeled leucine H3-leu), 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 3T3 cell 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 NPN1-1640 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, controlled temperature 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 the porous culture 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 one of following method to build up:
(a), to be selected from 199 substratum, the Eagle substratum, the RPN1-1640 substratum, the NEN substratum, the wherein a kind of of DNEN substratum is basic medium, with being selected from collagen gel, mouse 3T3 cell, human dermis inoblast and one of them trophoderm cultivated for epidermic cell that mixes fibroblastic collagen gel, in nutrient solution, add 5-20% calf serum or foetal calf serum, add and be selected from Urogastron, Toxins,exo-, cholera, the protein growth factor of Regular Insulin and be selected from hydrocortisone, 16-Methylprednisone acetate steroid hormone, to digest, filter and the epidermal basal cell suspension inoculation of centrifugal back acquisition in the nutrient solution of PH5-8, cultivate under controlled temperature 36-38 ℃;
(b), with selective medium NCDB153 substratum is basic medium, with being selected from collagen gel, mouse 3T3 cell, human dermis inoblast and the wherein a kind of trophoderm that has mixed fibroblastic collagen gel for the epidermic cell cultivation, add the pituitary body extract, add protein growth factor, be selected from Urogastron, Toxins,exo-, cholera, the protein growth factor of Regular Insulin and hydrocortisone, the hormone of 16-Methylprednisone acetate, to digest, filter and the epidermal basal cell suspension inoculation of centrifugal back acquisition in the nutrient solution of PH5-7.5, cultivate under controlled temperature 36-38 ℃;
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) in the epidermic cell culturing process, mix the biological active additive component of cosmetics that situation detects and screens by tritium-labeled nucleosides and tritium-labeled amino acid, 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 under the different biological active additive component of cosmetics 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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN97119496A CN1063226C (en) | 1997-12-02 | 1997-12-02 | Detecting and screening method for biological active additive component of cosmetics |
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| 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 CN1218837A (en) | 1999-06-09 |
| CN1063226C true CN1063226C (en) | 2001-03-14 |
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| CN97119496A Expired - Fee Related CN1063226C (en) | 1997-12-02 | 1997-12-02 | Detecting and screening method for biological active additive component of cosmetics |
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| CN107418996A (en) * | 2017-04-19 | 2017-12-01 | 中国食品药品检定研究院 | A kind of model for detecting vascular stent material Endothelialization prepares and application |
| 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 |
Citations (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 |
| CN1051758A (en) * | 1990-10-13 | 1991-05-29 | 哈尔滨市卫生防疫站 | Total number of bacterial colony detects glued membrane and preparation method |
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Patent Citations (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 |
| CN1051758A (en) * | 1990-10-13 | 1991-05-29 | 哈尔滨市卫生防疫站 | Total number of bacterial colony detects glued membrane and preparation method |
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| CN1218837A (en) | 1999-06-09 |
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