CN103908424A - Beautifying and nursing essence and preparation method thereof - Google Patents
Beautifying and nursing essence and preparation method thereof Download PDFInfo
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- CN103908424A CN103908424A CN201410074483.XA CN201410074483A CN103908424A CN 103908424 A CN103908424 A CN 103908424A CN 201410074483 A CN201410074483 A CN 201410074483A CN 103908424 A CN103908424 A CN 103908424A
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Abstract
The invention discloses a beautifying and nursing essence and a preparation method thereof. The essence is prepared from an epidermis cell nutrient solution and contains a plurality of cell growth factors, amino acids and vitamins. The essence mainly comprises an epidermal growth factor, a fibroblast growth factor, collagen type I, a stem cell growth factor, a horn cell growth factor and amino acids and vitamins including gamma-aminobutyric acid, coenzyme Q, medical vitamin C, medical vitamin B5 and medical vitamin E. The preparation method for the essence comprises the following steps: acquisition of epidermis of umbilical cord tissue of a newborn; cell culture; collection of a cell nutrient solution; filtration sterilization; condensation of a volume; addition of active components; addition of glycerin and hyaluronic acid; etc. The beautifying and nursing essence provides a plurality of endogenous nutritional ingredients with physiological activity for the skin, can promote metabolism of epidermis cells and repair damaged cells, has a moisture retention effect and is applicable to skin beautifying and nursing.
Description
Technical field
The present invention relates to a kind of biological cosmetics and preparation method thereof, be specifically related to a kind of human epidermal cell culture essence for cosmetology and preparation method thereof, belong to biotechnology and cosmetic field.
Background technology
In recent years, beauty treatment market emerges a large amount of facial treatment essence liquid.But the effective ingredient of existing essence mostly is plant component extraction thing, and liquid itself belongs to fine chemical product, and in order to improve the beautification function of cosmetics, majority contains the materials harmful to skin such as lead, hydrargyrum, ethanol, antiseptic, hormone, spice.Consumer, in using essence maintenance skin, has also caused very major injury to skin, even produces the problems such as mottle, red blood streak, allergy, has caused great threat to consumer is healthy.In addition, existing product is mostly refined and is made by chemical industry method, and the harmful organic substance in its product is residual more, and life-time service can have a negative impact to skin.And most existing products lack animality active substance, can not promote the metabolism of epidermis cell, improve skin quality.
Current disclosed a kind of fibroblast liquid for cosmetology, its main component is that fibroblast liquid contains cell culture fluid, collagen protein, cell growth factor, aminoacid and vitamin, and wherein cell growth factor comprises fibroblast growth factor, epithelical cell growth factor, hepatocyte growth factor and keratinocyte growth factor; The method that is prepared into fiber finer cytosol comprises to be obtained umbilical cord tissue hypodermal cell, former culture umbilical cord dermal tissue cell, goes down to posterity and cultivate umbilical cord tissue hypodermal cell, collecting cell culture fluid, add glycerol and cytokine to make the steps such as fibroblast liquid.Although this fibroblast liquid has the metabolic effect of the epidermis cell of promotion, cosmetology effect is limited.And this preparation method is easy, and easily culture fluid treatment step is few, the probability of microbial contamination is larger, therefore needs its processing technology to improve.
Cell culture essence, be the biological technical field derived product that recent years, new development was got up, utilize the biotic component in culture fluid, with classical skin-protection product glycerol, hyaluronic acid compatibility, make and there is bioactive beauty treatment essence, there is significant skin effect.And the production method of this product departed from Chemical Manufacture technique, unharmful substance is residual, has very high safety, has good promotion and application prospect.
Summary of the invention
In order to overcome the defect of processing technology of existing essence, the object of the present invention is to provide a kind of pure natural, without epidermis cell culture essence of organic substance residues and preparation method thereof.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
For an essence for cosmetology, this essence is made up of following component by weight percentage: the epidermal stem cells that adds multiple somatomedin and vitamin is cultivated concentrated solution 78.8%, glycerol 20% and hyaluronic acid 0.2%.
Further, described epidermal stem cells cultivation concentrated solution is that epidermal stem cells culture fluid is made according to the ratio of 5:1 is concentrated.
Further, described epidermal stem cells is cultivated in concentrated solution and has been added successively multiple somatomedin and vitamin.
Further, described multiple somatomedin and the contamination of vitamin are respectively: fibroblast growth factor 0.25 mg/L, epithelical cell growth factor 25.5 mg/L, NTx albumen 0.5 mg/L, keratinocyte growth factor 3 mg/L, ubiquinone 50g/L, medical vitamin C 50g/L, medical vitamin B
550g/L, medical vitamin E 50g/L.
For a preparation method for the essence of cosmetology, comprise the following steps:
Neonatal umbilical cord epidermis cell is obtained in step 1) collection: with the normal saline flushing neonatal umbilical cord tissue that contains mycillin, after being cut into small pieces, be layered in Tissue Culture Dish and cultivate;
Step 2) culture dish is coated: epidermis cell culture dish is coated with poly-D-lysine, naturally dries rear use;
Step 3) epidermis cell subculture: to adherent epidermis cell is carried out to former culture, change a subepidermal cell culture fluid for every three days by primary culture medium; Peptic cell, digestion finishes to stop digesting by the culture medium that goes down to posterity, in the cultivation of going down to posterity of 1:3 ratio;
Step 4) collecting cell culture fluid: collect the cell conditioned medium liquid before going down to posterity, obtain cell culture fluid;
Step 5) stock solution is filtered: the cell culture fluid of collection is through filtration system, elimination cell debris and microorganism;
Step 6) filtrate is concentrated: it is centrifugal that filtrate is placed in the super filter tube low-temperature and high-speed of 5K, and the concentrated solution obtaining is placed in 4 DEG C of refrigerator storage;
Step 7) adds cytokine: to the cytokine that adds accurate content in cell culture fluid;
Step 8) adds glycerol and hyaluronic acid: add medical glycerol and hyaluronic acid stock solution, mix homogeneously to adding above in the concentrated solution of cytokine.
Further, in described step 1), be at least 3 times with the number of times of the normal saline flushing neonatal umbilical cord tissue that contains mycillin, umbilical cord tissue is cut into the fritter of 0.2-0.5mm.
Further, described step 2) in the concentration of poly-D-lysine be 100ug/ml.
Further, described step 3) Central Plains culture base is NaHCO
3the DMEM in high glucose culture medium of content 0.37%, hyclone content 20%; The culture medium that goes down to posterity is NaHCO
3the DMEM in high glucose culture medium of content 0.37%, hyclone content 5%, peptic cell Digestive system used is 0.025% the pancreatin containing EDTA not, the condition of peptic cell is 37 DEG C, CO
2concentration 5%, 3 minute.
Further, in described step 4), collecting cell culture fluid is the cell culture fluid in cell 3-6 generation.
The filtration system of the cell culture fluid of further, collecting in described step 5) is the PES filtration system of aperture 0.22um.
Further, in described step 6), the centrifugal condition of low-temperature and high-speed is 4 DEG C of temperature, centrifugal force 12000 g, centrifugal 2 hours.
Further, the cytokine of adding in described step 7) is fibroblast growth factor 0.25 mg/L, epithelical cell growth factor 25.5 mg/L, NTx albumen 0.5 mg/L, keratinocyte growth factor 3 mg/L, ubiquinone 50g/L, medical vitamin C 50g/L, medical vitamin B
550g/L, medical vitamin E 50g/L.
Further, in described step 8), adding the final concentration of glycerol and hyaluronic acid stock solution is 20% and 0.2%.
The present invention has the following advantages and effect with respect to prior art:
1. the present invention adopts cell culture processes, obtains the culture fluid that contains multiple EGF, and the cytokine of these secretions is all human body component, can directly be absorbed by the skin, and avoids anaphylaxis, has no side effect.
2. the present invention adds the interpolation factor of multiple accurate content, and every kind of cytokine is all the endogenous cell factor of human body, has the function that promotes skin metabolism.A, fibroblast growth factor can promote fibroblast mitosis, mesoblastemic growth, also can stimulate vascularization, in wound healing and limb regeneration, play a role.B, epithelical cell growth factor can promote reparation and the regeneration of impaired epidermis, to burn, scald, phototherapy, etc. effect of reparation of multiple epidermis wound very remarkable.C, collagen protein are extracted by animal skins, containing a large amount of polar groups, is Moisture factor, has the tyrosine stoping in skin to be converted into melanic effect, therefore collagen protein has the effects such as pure natural moisturizing, whitening, wrinkle resistant, speckle dispelling, can be widely used in cosmetic products.D, keratinocyte growth factor all have stimulating growth splitting action to newborn or aging epithelial cell, have the significantly effect except cicatrix and Antiradiation injury.E, ubiquinone
10have the function that suppresses lipid peroxidation, its effect is than vitamin E, vitamin B
2more remarkable; can suppress mitochondrial peroxidating; the integrity of protection biofilm structure, has special potentiation to immunity, has the generation that improves cytophagous phagocytic rate, increase antibody, improves cell viability, removes free radical, improves the effects such as immunity of organisms, slow down aging.F, vitamin C promote the synthetic of collagen protein, if collagen protein lacks, can cause cell asynthesis.G, vitamin B
5horny layer is had to moisture-keeping function, improve skin coarseness, exfoliating skin, recover skin elasticity.H, vitamin E can be effectively to free radical resistings, LPO inhibitor, eliminating chloasma; The activity of restraint of tyrosinase, generates thereby reduce melanin.
3. cytokine is rich in the present invention, therefore can not use the mode of high temperature and chemosterilization.Therefore the present invention utilizes Filtration to remove microbial contamination, both avoided product to occur contamination and deterioration problem, ensure that again bioactive ingredients is not changed result by high temperature, radiation or chemical bactericide, ensured the activity of cytokine in product, make product effect stability.
4. the present invention has added glycerol and hyaluronic acid as basal liquid, has not only increased the moistening effect of essence of the present invention, has also increased the density of this liquid.
Brief description of the drawings
Accompanying drawing described herein is used to provide a further understanding of the present invention, forms the application's a part, and schematic description and description of the present invention is used for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is skin planarization improvement rate after the use essence of the embodiment of the present invention.
Fig. 2 is mottle skin lesion decline rate after the use essence of the embodiment of the present invention.
Specific embodiments
With specific embodiment, also the present invention is described in further detail by reference to the accompanying drawings below.
Embodiment 1: a kind of essence for cosmetology, this essence is made up of following following component by weight percentage: the epidermal stem cells that adds multiple somatomedin and vitamin is cultivated concentrated solution 78.8%, glycerol 20% and hyaluronic acid 0.2%.Its preparation method comprises the steps:
Neonatal umbilical cord epidermis cell is obtained in step 1) collection: with the normal saline flushing neonatal umbilical cord tissue that contains mycillin, the number of times of flushing is 3 times, then umbilical cord tissue is cut into after the fritter of 0.2-0.5mm and is layered in Tissue Culture Dish and cultivates;
Step 2) culture dish is coated: the poly-D-lysine that epidermis cell culture dish is 100ug/ml with concentration is coated with, and naturally dries rear use;
Step 3) epidermis cell subculture: to adherent epidermis cell is carried out to former culture, change a subepidermal cell culture fluid for every three days by primary culture medium; Peptic cell, digestion finishes to stop digesting by the culture medium that goes down to posterity, in the cultivation of going down to posterity of 1:3 ratio; Wherein: primary culture medium is NaHCO
3the DMEM in high glucose culture medium of content 0.37%, hyclone content 20%; The culture medium that goes down to posterity is NaHCO
3the DMEM in high glucose culture medium of content 0.37%, hyclone content 5%, peptic cell Digestive system used is 0.025% the pancreatin containing EDTA not, the condition of peptic cell is 37 DEG C, CO
2concentration 5%, 3 minute;
Step 4) collecting cell culture fluid: collect the cell conditioned medium liquid before the going down to posterity of 3-6 generation, obtain cell culture fluid;
Step 5) stock solution is filtered: the PES filtration system that the cell culture fluid of collection is 0.22um through aperture, elimination cell debris and microorganism;
Step 6) filtrate is concentrated: it is centrifugal that filtrate is placed in the super filter tube low-temperature and high-speed of 5K, and centrifugal condition is 4 DEG C of temperature, centrifugal force 12000 g, and centrifugal 2 hours, and make according to the ratio of 5:1 is concentrated, the concentrated solution obtaining is placed in 4 DEG C of refrigerator storage;
Step 7) adds cytokine: the cytokine to adding accurate content in cell culture fluid: fibroblast growth factor 0.25 mg/L, epithelical cell growth factor 25.5 mg/L, NTx albumen 0.5 mg/L, keratinocyte growth factor 3 mg/L, ubiquinone 50g/L, medical vitamin C 50g/L, medical vitamin B
550g/L, medical vitamin E 50g/L.；
Step 8) adds glycerol and hyaluronic acid: in above concentrated solution, add medical glycerol and hyaluronic acid stock solution, final concentration is respectively 20% and 0.2%, and mix homogeneously makes essence.
The present invention is for the efficient epidermal tissue's cell that obtains, and more effective technical scheme also has, and step 1) is cut into epidermal tissue the fritter of 0.2-0.5mm, so that epidermis cell departs from a large number from tissue.The condition of culture that the primary cell collection of step 1) is cultivated is: 37 DEG C, and CO
2concentration 5%, incubated overnight.
The present invention is in order to obtain a large amount of attached cells, and primary incubation time is 3-5 days, and condition of culture is 37 DEG C, CO
2concentration 5%.Primary culture medium is NaHCO
3the DMEM in high glucose of content 0.37%, hyclone content 20%.
The present invention is in order to ensure epidermal growth vigor, and Digestive system is 0.025% the trypsinization liquid without EDTA, and digestion condition is 37 DEG C, CO
2concentration 5%, 3 minute.When digestion finishes, stop digestion by the culture medium that goes down to posterity, the culture medium that goes down to posterity is NaHCO
3the DMEM in high glucose of content 0.37%, hyclone content 5%.
The present invention is in order to collect the culture fluid that cell growth factor content is high, collect the go down to posterity culture fluid of 3-6 of epidermis cell, now cell viability is vigorous, growth rapidly, in culture medium, a large amount of cytokines are secreted, and passage number too much can produce Toxic Metabolites, therefore collect the cell culture fluid in 3-6 generation.
The present invention, in order to increase skin moisture-keeping effect, increases the density of essence simultaneously, adds the medical glycerol of final concentration 20% in culture fluid, and the hyaluronic acid of final concentration 0.2%, makes the essence of certain density.
Embodiment 2: experiment effect test case, take following scheme:
Experimental subject: volunteer is from Shanghai, Suzhou, Wuxi, Jiangyin and Yixing in experiment, skin of face is due to acne, scytitis, cicatrix or old and feeble mottle and the wrinkle of producing.Experiment is rejected gestation, is intended gestation or breast-feeding female, rejects other dermatosis patients, as solar dermatitis, facial psoriasis, seborrheic dermatitis and urticaria, selects the age in 25-40 one full year of life, male's 42 examples, women's 68 examples.Volunteer forbids food edible pungent, that stimulate, smoking cessation, alleviating alcohol addiction at Clinical observation.Volunteer starts for 2 weeks from clinical, does not use hormone medicine and Claritin, does not use auxiliary skin care item.The experiment of volunteer's voluntary participation, signs Informed Consent Form.
Experimental technique: by professional dermatologist, volunteer's facial skin lesions is checked, take pictures.Volunteer sooner or later uses respectively this essence to be applied to face after clean.At the 7th, 14,28 days, respectively volunteer is followed up a case by regular visits to, and observe shooting, fill in clinical observation table.Adopt SAS6.12 software kit to data analysis.
Experimental result: 1. before treatment, only 1.2% the full face skin of volunteer is smooth, after treatment, the 7th, 14,28 days, the planarization improvement rate of the full face skin of volunteer after use essence as shown in Figure 1, was respectively: 1.2%, 9.8%, 11.5% and 25.1%.Show through Logistic regression analysis, use essence 28 days, skin planarization obviously improves, and before and after treatment, relatively, difference has statistical significance.2. use after this essence, after use essence, mottle skin lesion decline rate as shown in Figure 2, has statistical significance (P<0.05) with the front difference for the treatment of after treatment, shows that mottle skin lesion obviously improves.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (11)
1. for an essence for cosmetology, it is characterized in that: this essence by below by weight percentage component form: add multiple somatomedin and vitamin epidermal stem cells cultivate concentrated solution 78.8%, glycerol 20% and hyaluronic acid 0.2%.
2. a kind of essence for cosmetology according to claim 1, is characterized in that: it is that epidermal stem cells culture fluid is made according to the ratio of 5:1 is concentrated that described epidermal stem cells is cultivated concentrated solution.
3. a kind of essence for cosmetology according to claim 1, is characterized in that: described epidermal stem cells is cultivated in concentrated solution and added successively multiple somatomedin and vitamin.
4. a kind of essence for cosmetology according to claim 1, it is characterized in that: described multiple somatomedin and the contamination of vitamin are respectively: fibroblast growth factor 0.25 mg/L, epithelical cell growth factor 25.5 mg/L, NTx albumen 0.5 mg/L, keratinocyte growth factor 3 mg/L, ubiquinone 50g/L, medical vitamin C 50g/L, medical vitamin B
550g/L, medical vitamin E 50g/L.
5. the preparation method of a kind of essence for cosmetology claimed in claim 1, is characterized in that: comprise the following steps:
Neonatal umbilical cord epidermis cell is obtained in step 1) collection: with the normal saline flushing neonatal umbilical cord tissue that contains mycillin, after being cut into small pieces, be layered in Tissue Culture Dish and cultivate;
Step 2) culture dish is coated: epidermis cell culture dish is coated with poly-D-lysine, naturally dries rear use;
Step 3) epidermis cell subculture: to adherent epidermis cell is carried out to former culture, change a subepidermal cell culture fluid for every three days by primary culture medium; Peptic cell, digestion finishes to stop digesting by the culture medium that goes down to posterity, in the cultivation of going down to posterity of 1:3 ratio;
Step 4) collecting cell culture fluid: collect the cell culture supernatant before going down to posterity, obtain cell culture fluid;
Step 5) stock solution is filtered: the cell culture fluid of collection is through filtration system, elimination cell debris and microorganism;
Step 6) filtrate is concentrated: it is centrifugal that filtrate is placed in the super filter tube low-temperature and high-speed of 5K, and the concentrated solution obtaining is placed in 4 DEG C of refrigerator storage;
Step 7) adds cytokine: to the cytokine that adds accurate content in cell culture fluid;
Step 8) adds glycerol and hyaluronic acid: in above essence, add medical glycerol and hyaluronic acid stock solution, mix homogeneously.
6. the preparation method of a kind of essence for cosmetology according to claim 5, it is characterized in that: in described step 1), be at least 3 times with the number of times of the normal saline flushing neonatal umbilical cord tissue that contains mycillin, umbilical cord tissue is cut into the fritter of 0.2-0.5mm.
7. the preparation method of a kind of essence for cosmetology according to claim 5, is characterized in that: described step 2) in the concentration of poly-D-lysine be 100ug/ml.
8. the preparation method of a kind of essence for cosmetology according to claim 5, is characterized in that: described step 3) Central Plains culture base is NaHCO
3the DMEM in high glucose culture medium of content 0.37%, hyclone content 20%; The culture medium that goes down to posterity is NaHCO
3the DMEM in high glucose culture medium of content 0.37%, hyclone content 5%, peptic cell Digestive system used is 0.025% the pancreatin containing EDTA not, the condition of peptic cell is 37 DEG C, CO
2concentration 5%, 3 minute.
9. the preparation method of a kind of essence for cosmetology according to claim 5, is characterized in that: in described step 4), collecting cell culture fluid is the cell culture fluid in cell 3-6 generation.
10. the preparation method of a kind of essence for cosmetology according to claim 5, is characterized in that: the filtration system of the cell culture fluid of collecting in described step 5) is the PES filtration system of aperture 0.22um.
The preparation method of 11. a kind of essences for cosmetology according to claim 5, is characterized in that: in described step 6), the centrifugal condition of low-temperature and high-speed is 4 DEG C of temperature, centrifugal force 12000g, centrifugal 2 hours.
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| CN105055285A (en) * | 2015-08-26 | 2015-11-18 | 浙江奥瑞健生物技术有限公司 | Skin nutrition liquid and preparation method thereof |
| CN105168044A (en) * | 2015-09-01 | 2015-12-23 | 广州赛莱拉干细胞科技股份有限公司 | Collagen composition and collagen membrane sticker |
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