CN102552099A - Composite biological agent used for skin beautifying and restoring and preparing method - Google Patents
Composite biological agent used for skin beautifying and restoring and preparing method Download PDFInfo
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Abstract
The invention provides a composite biological agent used for skin beautifying and restoring and a preparing method. Active ingredients are extracted in cord blood stem cells of a puerperal, and active ingredients are extracted in placenta delivered by the puerperal, thus preparing the composite biological agent. The composite biological agent is used by the puerperal and can promote metabolism of stem cells on the surface of the puerperal, thus realizing the aims of beautifying, restoring, skin protection and scar and spot removal.
Description
Technical field
The present invention provides a kind of composite biological agent that beautifying skin is repaired that is used for, and is used for the beautifying skin reparation, and dispel scar speckle dispelling and puerpera's skin of skin protection recovers, and belongs to biological skin nursing technique field.
Background technology
At present, a lot of puerperas can follow problems such as facial colour spot, obesity, endocrine disturbance at phenolics, also can run into problems such as hyposexuality, recover of uterus obstacle, suckling potruncus are sagging, cyasma after the production.Cyasma is because the melanocyte-stimulating hormone of pregnancy period pituitary secretion increases, and a large amount of progestogen, estrogen, the increased functionality that causes the melanocyte in the skin so; Striae gravidarum is to increase along with the increase gradually of anemia of pregnant woman's gravid uterus and adrenal cortex can decompose the fibrinous glucocorticoid of elastic force in the pregnancy duration secretion; Make elastic fibrosis; Anemia of pregnant woman's abdominal skin tension force increases in addition; Make the elastorrhexis of skin, be the irregular level and smooth slightly striped of depression of volume purple or pale red.With advancing age, epidermal stem cells is fewer and feweri, so cyasma is difficult to lean on self epidermal stem cells recovery.
Correlational study shows, the cell ability that umbilical blood that puerpera self childbirth produces and Placenta Hominis have self renewal and differentiation potential can not only be in external height propagation, and also orientable inducing forms various types of human tissue cells.Placenta Hominis contains multiple bioactive substances such as hormone, cytokine, has antioxidation, and functions such as defying age and immunomodulating are precious medicine and health product raw materials, and Placenta Hominis is commonly called as afterbirth or afterbirth, and the traditional Chinese medical science is called Placenta Hominis.Its salty in the mouth, warm in nature, have benefiting qi and nourishing blood, the effect of benefit essence.Yet puerpera's Cord blood and Placenta Hominis of self when producing often but abandoned as refuse.
There are not problems such as joining type, ethics in the umbilical blood active component and the Placenta Hominis active component that produce according to puerpera self childbirth, and the anaphylaxis of avoiding the biological beauty product in external blood source to cause has good prospects for application and operability.
Described product contains the cord blood stem cell active component that parturient childbirth produces, and the stem cell active component content is 1-40%, contains the Placenta Hominis active component 3-15% that gives birth to when the puerpera produces.The present invention extracts the stem cell active component from puerpera self cord blood stem cell, from the Placenta Hominis that puerpera self gives birth to, extract the Placenta Hominis active component, is used for puerpera self.Promote the metabolism of trier's epidermal stem cells, thereby realize beauty treatment reparation, the dispel purpose of scar speckle dispelling of skin protection.The present invention also provides the method for preparing of above-mentioned composite biological agent.Contain the active component of the Placenta Hominis of puerpera's cord blood stem cell connector and puerpera self childbirth generation, belong to the medical cosmetology technical field.
Summary of the invention
The composite biological agent that the object of the present invention is to provide a kind of beautifying skin to repair, the umbilical cord and the Placenta Hominis that are produced by parturient childbirth are that feedstock production forms, and have autospecific and use, and have specificity, avoid advantages such as cross infection.
The present invention also provides the method for preparing of above-mentioned composite biological agent, is applicable to suitability for industrialized production.
Technical solution of the present invention is following:From puerpera self cord blood stem cell, extract the stem cell active component; From the Placenta Hominis that puerpera self gives birth to, extract the Placenta Hominis active component; Be configured to composite biological agent, be used for puerpera self and use, promote the metabolism of puerpera's epidermal stem cells; The purpose of scar speckle dispelling thereby realization beauty treatment reparation, skin protection are dispelled.
The composite biological agent that beautifying skin provided by the invention is repaired, mainly by following raw materials by weight portion than process (this paper abbreviation: active component of the present invention):
Cord blood stem cell active matter 3-15, stem cell active matter 1-40.
Above-mentioned composite biological agent is characterized in that:
Said stem cell active matter is from the umbilical blood that puerpera self childbirth produces, to extract.
Said Placenta Hominis active matter is from the Placenta Hominis that puerpera self childbirth produces, to extract.
The composite biological agent that the present invention is above-mentioned, also contain following material:
Fibroblast growth factor 0.05 ~ 0.1, epithelical cell growth factor 0.05 ~ 0.1, SOD3 ~ 7.
The method for preparing that is used for the composite biological agent of skin repair in puerperal disclosed by the invention may further comprise the steps:
One, the preparation of cord blood stem cell active matter:
1) cultivation of cord blood stem cell
Get healthy Cord blood and add 1 times of PBS dilution, blow and beat mixing, get the 50ml centrifuge tube and add the 10ml lymphocyte separation medium, slowly add the Cord blood of dilution, centrifugal 20 minutes of 2000rpm with suction pipe; With the slow sucking-off of the nebulous buffy coat of layering middle white, add 5ml PBS, centrifugal 5 minutes of 1000rpm abandons supernatant after centrifugal, adds 10ml PBS, mixing, repeated centrifugation is once; Abandon supernatant, add the α-MEM culture medium 20ml of 10% hyclone, mixing is placed on 100cm
2Culture bottle in, 37 ℃, 5%CO
2Cultivate;
2) passage amplification
When the cell of step 1) grew at the bottom of the 80-90% culture bottle, the sucking-off culture medium added 5ml PBS flushing, and sucking-off PBS adds the 2ml pancreatin; 37 ℃ of digestion 3 minutes adds α-MEM culture medium that 2ml contains 20% hyclone, in and pancreatin, the sucking-off neutralizer is put into the 50ml centrifuge tube; Add 20ml PBS, centrifugal 5 minutes of 1000rpm removes supernatant after centrifugal, adds 10ml PBS; Mixing, once centrifugal again, cell counting, every bottle adds 2 * 10
5Cell is inserted 100cm
2Culture bottle in, 37 ℃, 5%CO
2Cultivate;
3) preparation of stem cell active matter
With step 2) get the stem cell digestion counting 1 * 10 of cultivation
7Individual, PBS washing 3 times is dissolved in the 30ml normal saline ultrasonication cell, ultrasonic time 10 seconds, 5 seconds blanking times, ultrasound intensity 4, ultrasonic number of times 5; Make the abundant stripping of intracellular albumen and nutritional labeling, 4 ℃ centrifugal, gets supernatant after 1000rpm 10 minutes is centrifugal, removes cell debris; Ultrafilter membrane concentrates, and filter membrane aperture 50K removes immunoglobulin; Get filtrate, transfer to 30ml, aseptic with the membrane filtration of 0.22 μ m; Deciding concentration is 200 μ g/ml, and 4 ℃ of preservations get A liquid;
Get step 2) pH value of the survey culture medium used of cultured cell is 5.5 o'clock upper strata culture fluid 30ml, use the 30K ultrafiltration membrance filter, its volume is concentrated into about 5ml takes out, adding 5ml normal saline is settled to 10ml; With the membrane filtration of 0.22 μ m, make it aseptic, survey protein concentration, deciding concentration is 200 μ g/ml, 4 ℃ of preservations get B liquid;
In the 1:1 ratio A and B liquid are mixed, obtain the cord blood stem cell active matter;
Two, the preparation of Placenta Hominis active matter:
4) extracting of Placenta Hominis active substance
Get fresh human placenta with normal saline rinsing repeatedly, eliminate congestion, rub, the glycerol adding aqueous solution soaked 24 hours down in low temperature, hung filter, after getting the clear liquid adjust pH and being 4.5-5.5, added 0.5-12% Alumen, hold over night; Get supernatant, filtration fraction is collected in ultrafiltration, crosses the SephadexG-75 gel column, collects active component, filters once more, collects filtration fraction, uses the sterile glycerol aqueous solution to be diluted to concentration and is 20%-50%, extract, get C liquid;
5) preparation of protein hydrolyzate
The deposition of getting behind the step 4) liquid partly adds 6 times of weight distilled water, and adjust pH is to 7.8-8.1, and temperature is controlled at about 45 ℃, adds pancreatin; Enzyme dosage 1:250, enzymolysis 4 hours, every interval was got hydrolyzed solution in 1 hour and is surveyed amino acid content, heated and boiled; Transfer pH to 3, change in the pressure cooker, continued pyrohydrolysis 6 hours, pH to 6 is transferred in the cooling back; Sucking filtration is removed deposition, collects clear liquid and is protein hydrolyzate, gets D liquid;
Above-mentioned C liquid and D liquid are in 1: (2.5 ~ 3.5) ratio the Placenta Hominis active matter;
6) with the cord blood stem cell active matter of above-mentioned steps preparation, the Placenta Hominis active matter is mixed and made into the beautifying skin reparation in proportion with the inert matter of conventional cosmetics composite biological agent.
Said composite biological agent; The various forms that comprise injection, facial cream, emulsion, water liquid or cosmetics commonly used;, include but not limited to cleansing cream, cleansing soap, cleaning breast, facial film, astringent, massage cream, emulsion, nourishing cream, cosmetic cream, foundation cream or muffin etc. according to the cosmetics in all kinds of puerperal of the skin protection of purpose classification and beauty treatment.
The composite biological agent that described beautifying skin is repaired, mainly by following raw materials by weight portion than processing:
Cord blood stem cell active matter 3 ~ 15, stem cell active matter 1 ~ 40, culture medium 25 ~ 55,
Fibroblast growth factor 0.05 ~ 0.1, epithelical cell growth factor 0.05 ~ 0.1, SOD3 ~ 7.
The composite biological agent that described beautifying skin is repaired, by following raw materials by weight portion than processing:
Lanonol 3 ~ 5, cera alba 3 ~ 5, spermaceti 10 ~ 15, Borax 0.5 ~ 1.2, deionized water 50 ~ 80,
Navel blood stem cell active matter 3 ~ 15, Placenta Hominis active matter 1 ~ 40, culture medium 25 ~ 55, SOD3 ~ 7,
Fibroblast growth factor 0.05 ~ 0.1, epithelical cell growth factor 0.05 ~ 0.1.
The composite biological agent that described beautifying skin is repaired, by following raw materials by weight portion than processing:
Take by weighing lanonol 5g, cera alba 3g, spermaceti 10g; Borax 0.5g; Deionized water 50ml, navel blood stem cell active matter 15g, Placenta Hominis active matter 40g; Culture medium 55g, fibroblast growth factor 0.08g, epithelical cell growth factor 0.08g, SOD5g, its frost type cosmetics method preparation of producing by routine promptly gets.
The present invention relates to the purposes of above-mentioned composite biological agent product aspect beauty and skin care, comprise puerpera's reduce wrinkle in puerperal, whiten, speckle dispelling, reparation cyasma etc.
Metabolism is human life activity's a primitive form, and cell then is the ultimate unit of vital movement, and propagation of stem cell and differentiation have constituted tissue repair and regenerated basis.Therefore epidermal stem cells plays a significant role in skin metabolism.Through regulating the metabolism state of epidermal stem cells, can reach the effect of aspects such as skin-whitening, speckle dispelling, reparation cicatrix, this is the mechanism of action of this preparation just.
The stem cell composite factor can promote the propagation of epidermal stem cells; Promote the metabolism of skin, the Skin Cell vital movement strengthens, and the transportation of cellular material composition is frequent; Thereby promote the absorption of other beauty treatment compositions, this also is the superior difference with other products of this product.
Good effect of the present invention is: Skin Cell is had good facilitation, can accelerate cell metabolism, have the crinkle-removing whitening speckle dispelling effect of scar of dispelling, and can keep skin elasticity, it is blue and green naturally to recover skin; Simultaneously, because product of the present invention has reduced the anaphylactogen the biological product from root, make it possible to reduce to greatest extent the anaphylaxis of use crowd to cosmetics.Particularly the puerpera is had the good compatibility, be easier to be absorbed.
Description of drawings
Fig. 1, active component extracting solution of the present invention are to the influence of epidermis fibroblastic growth;
Fig. 2, the present invention are test-manufactured woman's wrinkle removing evaluation of effect of whitening;
Fig. 3, the present invention are test-manufactured the woman and are repaired cesarean cicatrix evaluation of effect;
Fig. 4 ~ Fig. 6, be experimenter of the present invention cesarean cicatrix and the striae gravidarum of the dispelling contrast photo of dispelling;
Fig. 7 ~ Figure 14 is a contrast photo before and after experimenter's speckle dispelling wrinkle removal of the present invention is whitened.
The specific embodiment:
Through following examples the present invention is described for example further; And do not limit the present invention in any way; Under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
Embodiment 1:
1,
The preparation of cord blood stem cell active matter
1) cultivation of cord blood stem cell:
Get Cord blood 5ml; Blood group chemical examination, record, five detections (hepatitis A, hepatitis B, hepatitis C, syphilis, AIDS) add PBS5ml, dilute 1 times, blow and beat mixing with suction pipe; Get the 50ml centrifuge tube and add the 10ml lymphocyte separation medium, slowly add the stem cell suspension of dilution; Centrifugal 20 minutes of 2000rpm; The visible obviously nebulous one deck of layering middle white in centrifugal back is a buffy coat, with its slow sucking-off, adds 5ml PBS, and centrifugal 5 minutes of 1000rpm abandons supernatant after centrifugal, adds PBS10ml, mixing, and repeated centrifugation is once; Abandon supernatant, add the α-MEM culture medium 20ml of 10% hyclone, mixing is placed on 100cm
2Culture bottle in, 37 ℃, 5%CO
2Cultivate.
2) passage amplification:
When cell grew at the bottom of the 80-90% culture bottle, the sucking-off culture medium added the 5mlPBS flushing, and sucking-off PBS adds the 2ml pancreatin; 37 ℃ of digestion 3 minutes adds 2ml and contains in the α-MEM culture medium of 20% hyclone and pancreatin, and the sucking-off neutralizer is put into the 50ml centrifuge tube; Add 20mlPBS, centrifugal 5 minutes of 1000rpm removes supernatant after centrifugal, adds PBS10ml; Mixing, once centrifugal again, cell counting, every bottle adds 2 * 10
5Cell is inserted 100cm
2Culture bottle in, 37 ℃, 5%CO
2Cultivate.
3) preparation of stem cell active component:
With stem cell digestion counting 1 * 10
7 PBS washing 3 times is dissolved in the 30ml normal saline ultrasonication cell: ultrasonic time 10 seconds, 5 seconds blanking times, ultrasound intensity 4, ultrasonic number of times 5; Make the abundant stripping of intracellular albumen and nutritional labeling, 4 ℃ centrifugal, got supernatant after centrifugal, and removed cell debris in 1000rpm10 minute; Ultrafilter membrane concentrates, and filter membrane aperture 50K removes immunoglobulin, gets filtrate; Transfer to 30ml,, make it aseptic with the membrane filtration of 0.22 μ m; Deciding concentration is 200 μ g/ml, and 4 ℃ of preservations are labeled as A liquid.
Cell is long when the most vigorous, and surveying culture medium (IMDM) pH value is 5.5, gets upper strata culture fluid 30ml, use the 30K ultrafiltration membrance filter, its volume is concentrated into about 5ml takes out, and adding 5ml normal saline is settled to 10ml; With the membrane filtration of 0.22 μ m, make it aseptic, survey protein concentration, deciding concentration is 200 μ g/ml, 4 ℃ of preservations are labeled as B liquid.
In the 1:1 ratio above-mentioned A and B liquid are mixed, obtain the cord blood stem cell active matter.
, Placenta Hominis bioactive substance method for preparing:
1) extracting of active substance:
Get fresh human placenta, eliminate congestion with the normal saline rinsing, rub, the glycerol adding aqueous solution soaked 24 hours down in low temperature, hung filter, after getting clear liquid and transferring pH value, added 0.5-12% Alumen, hold over night.Get supernatant, at 4 atmospheric pressure 1cm
2Filtration fraction is collected in following ultrafiltration, crosses the SephadexG-75 gel column, collects active component, filters once more, collects filtration fraction, with the sterile glycerol aqueous solution be diluted to finite concentration extract, be referred to as c liquid.
2) preparation of protein hydrolyzate:
Get above-mentioned deposition part of hanging behind the liquid, adopt enzyme-sour associating hydrolysis.Get deposition and add 6 times of weight distilled water, transfer pH value to alkalescence, temperature is controlled at about 45 ℃, adds pancreatin; Enzyme dosage 1:250. enzymolysis 4 hours, every interval were got hydrolyzed solution in 1 hour and are surveyed amino acid content, heated and boiled, and transferred about PH to 3; Change in the pressure cooker, continued pyrohydrolysis again 6 hours, transfer about PH to 6 the cooling back; Sucking filtration is removed deposition, collects clear liquid and is protein hydrolyzate, is referred to as d liquid.
Above-mentioned C liquid and D liquid are in 1: (2.5 ~ 3.5) ratio the Placenta Hominis active matter.
Take by weighing lanonol 5g, cera alba 3g, spermaceti 10g; Borax 0.5g; Deionized water 50ml, navel blood stem cell active matter 15g, Placenta Hominis active matter 40g; Culture medium (IMDM) 55g, fibroblast growth factor 0.08g, epithelical cell growth factor 0.08g, SOD5g, its frost type cosmetics method preparation of producing by routine promptly gets.
Take by weighing lanonol 3g, cera alba 3g, spermaceti 15g; Borax 0.8g; Deionized water 70g, navel blood stem cell active matter 10g, Placenta Hominis active matter 25g; Culture medium (IMDM) 40g, fibroblast growth factor 0.1g, epithelical cell growth factor 0.1g, SOD 3g, its frost type cosmetics method preparation of producing by routine promptly gets.
Take by weighing lanonol 5g, cera alba 5g, spermaceti 15g; Borax 1.2g, deionized water 80g, navel blood stem cell active matter 10g; Placenta Hominis active matter 10g; Culture medium (IMDM) 25g, fibroblast growth factor 0.05g, epithelical cell growth factor 0.05g, SOD 7g,, its frost type cosmetics method preparation of producing by routine promptly gets.
Take by weighing lanonol 5g, cera alba 4g, spermaceti 10g; Borax 1.0g; Deionized water 50g, navel blood stem cell active matter 15g, Placenta Hominis active matter 25g; Culture medium (IMDM) 40g, fibroblast growth factor 0.1g, epithelical cell growth factor 0.1g, SOD 5g, its frost type cosmetics method preparation of producing by routine promptly gets.
Test Example 1
One, active component of the present invention is to the influence of fibroblastic growth
Embodiment: SF is by 1 * 10
4/ hole is inoculated in 96 orifice plates, and 6 multiple holes inoculations are divided into: five groups of blank group a, culture medium negative control group b, stem cell active component group c, Placenta Hominis active component d group, preparation group e of the present invention.Inoculate the 7 96 identical culture plates in hole altogether, observed 7 days, detect a plate every day.The blank group adds PBS liquid 200 μ l, and negative control group is the fibroblast culture fluid, and the stem cell active matter and the Placenta Hominis active matter of embodiment 1 preparation all add in the culture fluid in the 3:1 ratio.Began in second day to detect, first plate adds MTT20 μ l/ hole (5mg/ml), adds DMSO after 4 hours, surveys cell growth number, the upgrowth situation of observation of cell.Fibroblast culture fluid wherein is the α-MEM culture medium that contains 10% hyclone.Referring to Fig. 1; As can beappreciated from fig. 1; The simple b line of navel blood stem cell active component that uses is faster than d line with the cell proliferation rate of the c line representative of simple use Placenta Hominis active component; The cell proliferation rate of a line representative of Combined application navel blood stem cell active component and Placenta Hominis active component is the fastest, approximately is 2 times of negative control group.
Conclusion:Adding stem cell active component of the present invention and Placenta Hominis active component plays a driving role to the growth of SF; The metabolism of cell has been accelerated in this effect; To the rejuvenation of the skin important function of getting up; Active component just of the present invention has better reparation SF, the effect of delaying aging.
Two, the stability test of active component of the present invention:
Get and take a sample after composite biological agent of the present invention places 37 ℃ of incubators and 4 ℃ of refrigerators and room temperature to place different time respectively respectively, carry out determination of activity.
Embodiment: SF is by 1 * 10
4/ hole is inoculated in 96 orifice plates, and 6 multiple holes inoculations are divided into: blank group, culture medium negative control group, active component of the present invention are respectively five groups of 37 ℃ of incubator preservation groups, 4 ℃ of refrigerator preservation groups and room temperature preservation groups; Co-cultivation 7 days.The blank group adds PBS liquid 200 μ l, and negative control group is the fibroblast culture fluid, and the stem cell active component of embodiment 1 preparation and Placenta Hominis active component all add in the culture fluid in the 3:1 ratio; Began in the 8th day to detect, add MTT20 μ l/ hole (5mg/ml), add DMSO after 4 hours, survey cell growth number, the upgrowth situation of observation of cell.Fibroblast culture fluid wherein is the α-MEM culture medium that contains 10% hyclone.
The ratio to the influence of fibroblastic growth propagation that active component of the present invention is cultivated 7 days with the different resting periods of different temperatures to the influence and the preservation in the 0th day of fibroblastic growth propagation altogether serves as to detect foundation.The result sees table 1.
Can find out that from table 1 active component of the present invention is when 37 ℃, 4 ℃, room temperature held to 180 day, its activity is not seen significant change.The result shows that this present invention active component can reach 180 days in room temperature and 4 ℃ of following preservations, as it is more excellent to place 37 ℃ of incubators to preserve.
Test Example 2
With the composite biological agent of embodiment 2 ~ 5 preparation wrinkle removing contrast test of whitening, observe its zest anaphylaxis and untoward reaction simultaneously to skin.
Adopt single blind method, promptly the experimenter is ignorant to product composition, will be test-manufactured woman's random packet, is respectively common facial cream group (a), adds navel blood stem cell active component group (b), adds Placenta Hominis active component group (c), preparation group of the present invention (d).Every group 10 people; Experimenter every day, face was smeared preparation of the present invention 2 times sooner or later, the product effect was once estimated in per 3 days, and the wrinkle removing comprehensive grading of whitening is divided into Three Estate and is respectively DeGrain (0-3 branch); Effect general (4-6 branch), effect is (7-10 branch) significantly.After one month general effect is carried out the mark evaluation, the result sees Fig. 2.
Conclusion: skin changes noticeably before and after as can beappreciated from fig. 2 using the facial cream that adds navel blood stem cell active component and Placenta Hominis active component, uses the back wrinkle obviously to reduce, and skin of face bleaches, and skin colour brightens.Do not find that this compound formulation has zest to skin, have that 1 people has slight allergy, the phenomenon of itching.
Test Example 3
Composite biological agent with embodiment 2 ~ 5 preparations is repaired cesarean cicatrix contrast test, observes its zest anaphylaxis and untoward reaction to skin simultaneously.
Adopt single blind method, promptly the experimenter is ignorant to product composition, will be test-manufactured woman's random packet, is respectively simple nutritional solution group (a), adds navel blood stem cell active component group (b), adds Placenta Hominis active component group (c), preparation group of the present invention (d).Every group 10 people; The experimenter sooner or later smears preparation of the present invention 2 times at the cesarean edge of a knife position every day; The product effect once estimated in per 3 days, cicatrix is repaired comprehensive grading and is divided into Three Estate and is respectively DeGrain (0-3 branch), effect general (4-6 branch); Effect is (7-10 branch) significantly, after one month general effect is carried out the mark evaluation.Fig. 3 ~ Fig. 6 is dispel the cesarean cicatrix and the striae gravidarum contrast photo of dispelling of selecting 3 triers.
Conclusion: the present invention can be used for puerpera self and use, and promotes the metabolism of puerpera's epidermal stem cells, thereby realizes beauty treatment reparation, the dispel purpose of scar speckle dispelling of skin protection.
Test Example 4
Use the composite biological agent of the embodiment of the invention 2 ~ 5 preparations to carry out the speckle dispelling wrinkle removal cosmetic result contrast test of whitening, observe its zest anaphylaxis and untoward reaction simultaneously skin.
Selected volunteer's 80 examples, the experimenter sooner or later smears preparation of the present invention 2 times by the skin protection method face of routine every day, and duration of trial is 3 months, effective 72 examples of statistics conclusion; Produce effects 6 examples; Invalid 2 examples do not find that compound formulation of the present invention has zest to skin.Fig. 7 ~ Figure 14 is a contrast photo before and after the speckle dispelling wrinkle removal of selecting 8 triers is whitened;
Conclusion: use the speckle wrinkle and the whitening effect of preparation of the present invention front and back skin to change noticeably, have good speckle dispelling wrinkle removal whitening function.
Claims (8)
1. the composite biological agent repaired of a beautifying skin, mainly process by ratio of weight and the number of copies by following material:
Cord blood stem cell active matter 3 ~ 15, Placenta Hominis active matter 1 ~ 40.
2. the composite biological agent above-mentioned like claim 1 is characterized in that:
Said cord blood stem cell active matter is from the umbilical blood that puerpera self childbirth produces, to extract;
Said Placenta Hominis active matter is from the Placenta Hominis that puerpera self childbirth produces, to extract.
3. the composite biological agent above-mentioned like claim 1 is characterized in that also containing following material:
Fibroblast growth factor 0.05 ~ 0.1, epithelical cell growth factor 0.05 ~ 0.1, SOD3 ~ 7.
4. like the method for preparing of right 1 or 2 said composite biological agents, may further comprise the steps:
The preparation of cord blood stem cell active matter:
1) cultivation of cord blood stem cell
Get healthy Cord blood and add 1 times of PBS dilution, blow and beat mixing, get the 50ml centrifuge tube and add the 10ml lymphocyte separation medium, slowly add the Cord blood of dilution, centrifugal 20 minutes of 2000rpm with suction pipe; With the slow sucking-off of the nebulous buffy coat of layering middle white, add 5ml PBS, centrifugal 5 minutes of 1000rpm abandons supernatant after centrifugal, adds 10ml PBS, mixing, repeated centrifugation is once; Abandon supernatant, add the α-MEM culture medium 20ml of 10% hyclone, mixing is placed on 100cm
2Culture bottle in, 37 ℃, 5%CO
2Cultivate;
2) passage amplification
When the cell of step 1) grew at the bottom of the 80-90% culture bottle, the sucking-off culture medium added 5ml PBS flushing, and sucking-off PBS adds the 2ml pancreatin; 37 ℃ of digestion 3 minutes adds α-MEM culture medium that 2ml contains 20% hyclone, in and pancreatin, the sucking-off neutralizer is put into the 50ml centrifuge tube; Add 20ml PBS, centrifugal 5 minutes of 1000rpm removes supernatant after centrifugal, adds 10ml PBS; Mixing, once centrifugal again, cell counting, every bottle adds 2 * 10
5Cell is inserted 100cm
2Culture bottle in, 37 ℃, 5%CO
2Cultivate;
3) preparation of stem cell active matter
With step 2) get the stem cell digestion counting 1 * 10 of cultivation
7Individual, PBS washing 3 times is dissolved in the 30ml normal saline ultrasonication cell, ultrasonic time 10 seconds, 5 seconds blanking times, ultrasound intensity 4, ultrasonic number of times 5; Make the abundant stripping of intracellular albumen and nutritional labeling, 4 ℃ centrifugal, gets supernatant after 1000rpm 10 minutes is centrifugal, removes cell debris; Ultrafilter membrane concentrates, and filter membrane aperture 50K removes immunoglobulin; Get filtrate, transfer to 30ml, aseptic with the membrane filtration of 0.22 μ m; Deciding concentration is 200 μ g/ml, and 4 ℃ of preservations get A liquid;
Get step 2) pH value of the survey culture medium used of cultured cell is 5.5 o'clock upper strata culture fluid 30ml, use the 30K ultrafiltration membrance filter, its volume is concentrated into about 5ml takes out, adding 5ml normal saline is settled to 10ml; With the membrane filtration of 0.22 μ m, make it aseptic, survey protein concentration, deciding concentration is 200 μ g/ml, 4 ℃ of preservations get B liquid;
In the 1:1 ratio above-mentioned A and B liquid are mixed, obtain the cord blood stem cell active matter;
The preparation of Placenta Hominis active matter:
4) extracting of Placenta Hominis active substance
Get fresh human placenta with normal saline rinsing repeatedly, eliminate congestion, rub, the glycerol adding aqueous solution soaked 24 hours down in low temperature, hung filter, after getting the clear liquid adjust pH and being 4.5-5.5, added 0.5-12% Alumen, hold over night; Get supernatant, filtration fraction is collected in ultrafiltration, crosses the SephadexG-75 gel column, collects active component, filters once more, collects filtration fraction, uses the sterile glycerol aqueous solution to be diluted to concentration and is 20-50%, extract, get C liquid;
5) preparation of protein hydrolyzate
The deposition of getting behind the step 4) liquid partly adds 6 times of weight distilled water, and adjust pH is to 7.8-8.1, and temperature is controlled at about 45 ℃, adds pancreatin; Enzyme dosage 1:250, enzymolysis 4 hours, every interval was got hydrolyzed solution in 1 hour and is surveyed amino acid content, heated and boiled; Transfer pH to 3, change in the pressure cooker, continued pyrohydrolysis 6 hours, pH to 6 is transferred in the cooling back; Sucking filtration is removed deposition, collects clear liquid and is protein hydrolyzate, gets D liquid;
Above-mentioned C liquid and D liquid are in 1: (2.5 ~ 3.5) ratio the Placenta Hominis active matter;
6) with the cord blood stem cell active matter of above-mentioned steps preparation, the Placenta Hominis active matter is mixed and made into the beautifying skin reparation in proportion with the inert matter of conventional cosmetics composite biological agent.
5. composite biological agent according to claim 1; The various forms that comprise injection, facial cream, emulsion, water liquid or cosmetics commonly used;, include but not limited to cleansing cream, cleansing soap, cleaning breast, facial film, astringent, massage cream, emulsion, nourishing cream, cosmetic cream, foundation cream or muffin etc. according to the cosmetics in all kinds of puerperal of the skin protection of purpose classification and beauty treatment.
6. the composite biological agent that beautifying skin as claimed in claim 1 is repaired, mainly by following raw materials by weight portion than processing:
Cord blood stem cell active matter 3 ~ 15, stem cell active matter 1 ~ 40, culture medium 25 ~ 55,
Fibroblast growth factor 0.05 ~ 0.1, epithelical cell growth factor 0.05 ~ 0.1, SOD3 ~ 7.
7. the composite biological agent that beautifying skin as claimed in claim 1 is repaired, by following raw materials by weight portion than processing:
Lanonol 3 ~ 5, cera alba 3 ~ 5, spermaceti 10 ~ 15, Borax 0.5 ~ 1.2, deionized water 50 ~ 80,
Navel blood stem cell active matter 3 ~ 15, Placenta Hominis active matter 1 ~ 40, culture medium 25 ~ 55, SOD3 ~ 7,
Fibroblast growth factor 0.05 ~ 0.1, epithelical cell growth factor 0.05 ~ 0.1.
8. the composite biological agent that beautifying skin as claimed in claim 1 is repaired, by following raw materials by weight portion than processing:
Take by weighing lanonol 5g, cera alba 3g, spermaceti 10g; Borax 0.5g; Deionized water 50ml, navel blood stem cell active matter 15g, Placenta Hominis active matter 40g; Culture medium 55g, fibroblast growth factor 0.08g, epithelical cell growth factor 0.08g, SOD5g, its frost type cosmetics method preparation of producing by routine promptly gets.
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