CN110200839A - The application of big rat-tail Type I collagen albumen - Google Patents
The application of big rat-tail Type I collagen albumen Download PDFInfo
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- CN110200839A CN110200839A CN201910520678.5A CN201910520678A CN110200839A CN 110200839 A CN110200839 A CN 110200839A CN 201910520678 A CN201910520678 A CN 201910520678A CN 110200839 A CN110200839 A CN 110200839A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
- A61K8/988—Honey; Royal jelly, Propolis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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Abstract
The invention discloses the applications of big rat-tail Type I collagen albumen.The big rat-tail Type I collagen albumen of the present invention is applied in the product for preparing wrinkle resistant moisturizing and/or whitening.The wrinkle resistant moisturizing of the big rat-tail Type I collagen albumen of the present invention, whitening product are more advantageous to the migration of human skin fibroblasts, it was demonstrated that product of the present invention are conducive to the wrinkle resistant moisturizing of skin.The big rat-tail Type I collagen albumen of the present invention has to TYR enzyme activity inhibition, it was demonstrated that it is with whitening function.The present invention is big, and rat-tail Type I collagen albumen has the characteristics that safe without toxic side effect.
Description
Technical field
The present invention relates to the applications of big rat-tail Type I collagen albumen, and in particular to a kind of rat-tail Type I collagen albumen is beautiful in preparation
Application and a kind of cosmetics in white Wrinkle-eliminating cosmetics, belong to cosmetic field.
Background technique
Rat-tail Type I collagen albumen (Collagen from rat tail, Type I) system uses the side Birkedal-Hansen
Method reaches 95% or more in sterile lower preparation, purity, dissolves in 0.006mol/L acetic acid.This product can be used for the packet of Tissue Culture Dish
Quilt is particularly suitable for the culture that ordinary cells culture vessel is not easy attached cell；It can also be used for preparing three-dimensional collagen gel, make cell
It is grown in the three-dimensional environment of simulation.It is widely applied about in cell culture.
Peony extract is rich in natural anti-oxidation active constituent, protects damage of the skin by exogenous free radical.
Propolis extract is rich in Immunity active factor, enhances the immunity of skin, prevents skin infection.
Summary of the invention
The object of the present invention is to provide the applications of big rat-tail Type I collagen albumen, specifically provide a kind of rat-tail Type I collagen albumen
Application in preparation whitening Wrinkle-eliminating cosmetics.
Big rat-tail Type I collagen albumen provided by the invention is applied in the product for preparing wrinkle resistant moisturizing and/or whitening.
In above-mentioned application, the product includes at least one of food, cosmetics and health care product.
The present invention also provides the cosmetics of a kind of wrinkle resistant moisturizing and/or whitening, active constituent is big I type of rat-tail
Collagen.
In the present invention, the preparation method of the big rat-tail Type I collagen albumen includes the following steps:
1, rat tail is taken to clean, 75% alcohol impregnates 5 minutes；
2, tail is cut off, removes fur by, and is cut into segment, extracts the tail key of silver color out；
3, tail tendon cutting is placed in plate, PBS (pH7.2~7.4, KH2PO42mM, Na2HPO48mM, NaCl
136mM, KCl 2.6mM) it impregnates；
4, all tail keys are put in the Tris-HCl of 0.05mol/L, 4 DEG C overnight；
5, Tris-HCl is sucked, tail tendon is weighed into (0.5-1 grams)；
6, tail tendon is placed in plate and is shredded, is transferred in conical flask；
7, in the ratio of every gram of tail tendon 50mL, 0.1% acetum is added；
8, it rocks, is scattered in tail tendon in acetum, 4 DEG C of one weeks of placement；
9, it is centrifuged, 4 degree 4000 revs/min, 30 minutes；
10, supernatant is drawn, 300 mesh filters are crossed；
11,100ml 0.14mol/LNaOH (molten to 1000 milliliters water of 5.6gNaOH are added in the above-mentioned crude extract of every 600ml
In), 8000 turns of 5min centrifugations abandon supernatant, stay flocculent deposit；
12, flocky precipitate is placed in tri-distilled water, with 0.1mol/L (1000:6) acetate dissolution sediment.
The present invention also provides a kind of cosmetics, which includes that the component of following mass parts is made: I type glue of big rat-tail
2~3 parts of former albumen；0.5~1 part of peony extract；0.6~1.2 part of propolis extract；3 parts of Nexbase 2004；Caprylic capric
4 parts of glyceryl ester；3 parts of dimethicone；1.2 parts of cetanol；0.5 part of docosyl alcohol；4 parts of different tridecanol isononoate；Water 100
Part；3 parts of glycerol；3 parts of butanediol；1 part of glycerol stearate (SE)；- 20 1.5 parts of docosyl alcohol polyethers；Disodium ethylene diamine tetraacetate
0.03 part；0.5 part of copolymer of sodium acrylate acyl dimethyltaurine sodium；0.2 part of preservative；0.1~0.3 part of essence.
In the present invention, the peony extract and the propolis extract be extract well known in the art or according to
Method well known in the art is extracted.
Invention further provides the preparation method of above-mentioned cosmetics, include the following steps: Nexbase 2004, pungent 1)
Sour capric acid glyceryl ester, dimethicone, cetanol, docosyl alcohol and the mixing of different tridecanol isononoate, heating obtain A phase；
2) big rat-tail Type I collagen albumen, peony extract water, glycerol, butanediol, glycerol stearate, docosyl alcohol are gathered
Ether -20 and disodium ethylene diamine tetraacetate are mixed to get B phase, and then the A obtained in step 1) is added in the B phase,
Pre-emulsification, homogeneous；
3) through A phase described in step 2) with the B phase after processing, be added sodium acrylate acyl dimethyltaurine sodium copolymerization
Object mixing；
4) step 3) treated system is cooling, preservative, essence homogeneous is added then to get the cosmetics are arrived.
Above-mentioned preparation method, it is described to be heated to 80 DEG C in step 1)；
In step 2), the pre-emulsified time is 5min, and the time of the homogeneous is 3min；
In step 3), the system is cooled to 45 DEG C；
In step 4), the time of the homogeneous is 2min.
The invention has the following advantages that
The big rat-tail Type I collagen albumen of the present invention and its formula cosmetics of preparation have effects that wrinkle resistant moisturizing, whitening, more
Be conducive to the migration of human skin fibroblasts, it was demonstrated that product of the present invention are conducive to the wrinkle resistant moisturizing of reparation of skin.Rat of the present invention
Tail Type I collagen albumen has to TYR enzyme activity inhibition, it was demonstrated that it is with whitening function.The big I type glue of rat-tail of the present invention
The acute skin irritation of former albumen is tested, acute ocular irritation test, skin phototoxicity test, Skin allergy test,
Mammalian cells in vitro chromosomal aberration test is showed no toxic reaction.The present invention is big, and rat-tail Type I collagen albumen is orally acute
Toxicity test MTD is all larger than 15.0g/kgBW；Genetic toxicity test: Ames test, the examination of mouse bone marrow polychromatic erythrocytes micronucleus
It tests and is showed no mutagenesis；Feeding test for 90 days is had no as the result is shown to the weight of animals, food utilization, hematology, blood
There are adverse effect in biochemical, dirty body ratio and histopathologic examination.The big rat-tail Type I collagen albumen of the present invention has safe and non-toxic secondary work
The characteristics of using.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In following embodiments, the preparation method of big rat-tail Type I collagen albumen includes the following steps:
1, rat tail is taken to clean, 75% alcohol impregnates 5 minutes；
2, tail cut off, remove fur, and be cut into segment, extract the tail key of silver color out；
3, tail tendon cutting is placed in plate, PBS (pH7.2~7.4, KH2PO42mM, Na2HPO48mM, NaCl
136mM, KCl 2.6mM) it impregnates；
4, all tail keys are put in the Tris-HCl of 0.05mol/L, 4 DEG C overnight；
5, Tris-HCl is sucked, tail tendon is weighed into (0.5-1 grams)；
6, tail tendon is placed in plate and is shredded, is transferred in conical flask；
7, in the ratio of every gram of tail tendon 50mL, 0.1% acetum is added；
8, it rocks, is scattered in tail tendon in acetum, 4 DEG C of one weeks of placement；
9, it is centrifuged, 4 degree 4000 revs/min, 30 minutes；
10, supernatant is drawn, 300 mesh filters are crossed；
11,100ml 0.14mol/LNaOH (molten to 1000 milliliters water of 5.6gNaOH are added in the above-mentioned crude extract of every 600ml
In), 8000 turns of 5min centrifugations abandon supernatant, stay flocculent deposit；
12, flocky precipitate is placed in tri-distilled water, with 0.1mol/L (1000:6) acetate dissolution sediment.
Embodiment 1, formula cosmetics, the wrinkle resistant moisturizing of big rat-tail Type I collagen albumen, whitening function experiment
Be formulated cosmetics: active constituent is rat-tail I-type collagen, and peony extract, propolis extract have and protect
Wet, wrinkle resistant, whitening function, specific formula be table 1 shown in,
Table 1
It is formulated the preparation method of cosmetics, specifically comprises the following steps: that each component in A phase is added in container A respectively, adds
Heat is to 80 DEG C；Each component in B phase is added in container B simultaneously, is heated to 80 DEG C；While stirring B phase, A is added to, in advance
5min is emulsified, then homogeneous 3min；While agitating, C phase component is added；Cooling down is stirred, is cooled to 45 to product
After DEG C, D phase each component, homogeneous 2min is added；Continue stirring cooling, drops to room temperature.
One, the wrinkle resistant moisture-keeping functions replication experiment of the skin of big rat-tail Type I collagen albumen:
1, materials and methods
1.1 key instrument equipment
Experiment is shown in Table 2 with key instrument equipment.
The experiment key instrument equipment list of table 2
1.2 main agents
Main agents are shown in Table 3.
Table 3 predominantly detects reagent list
1.3 experimental method
1.3.1 collagen bed board:
Big rat-tail Type I collagen albumen, ox bone Type I collagen albumen, the sterile 0.006mol/L acetic acid of fish-skin albumen
(0.36g/L) will be diluted to 0.1mg/mL respectively.On the super-clean bench with 0.22 μm of filtering with microporous membrane degerming, then it is sub-packed in
In EP pipe.With evening before that day bed board-be rule of thumb by 5 μ g/cm2Density, be placed in disinfection super-clean bench overnight (most
It is 18 hours or more well, the time, too short collagen was difficult to really attach), it dries.It is rinsed 2 times with sterile PBS liquid, then trained with cell
Nutrient solution is rinsed 1 time, last inoculating cell.
1.3.2 cell is passed on and is proliferated:
With the DMEM-H culture medium containing 1% mycillin of 10% fetal calf serum of concentration expressed in percentage by volume and volumn concentration,
Put 37 DEG C of people, volumn concentration 5%CO2: cell incubator in cultivated.Collect rate to cell and reaches culture plate bottom surface
When 90% or more product, using 0.25% trypsin digestion, secondary culture is carried out.Take the 3rd~6 generation cell for follow-up study.
1.3.3 scratch experiment:
Scratch experiment utilizes inverted microscope observation cell growth status and metamorphosis.Scarification detects different collagens pair
The influence of human fibroblasts migration takes the 3rd~6 generation human fibroblasts, is inoculated in big rat-tail Type I collagen albumen, I type of ox bone
In 12 orifice plates that collagen, fish-skin albumen embed, routine culture discards culture when cell collects rate and reaches 95% or more
Base, serum free medium starvation cultivate 12~16h；Using 10 microlitres of suction pipette heads, ruler contrasts scratch, and PBS washing falls off
Cell counts scratch inner cell number under 10 times of mirrors, for 24 hours after measure cell number in scratch again, every group takes 6 visuals field at random,
For later statistical analysis.Experiment is repeated 3 times.
All results of 1.4 statistical procedures are all made of 13.0 software of SPSS and carry out statistical analysis.Experimental result usesIt indicates, group difference uses one-way analysis of variance, and P < 0.05 is that difference has statistical significance.
2 results
2.1 human fibroblasts morphological observation inverted microscopes observe cellular morphology, after scratch experiment 24 hours, this hair
Six orifice plate scratch inner cell quantity of bright product processing are significantly greater than ox bone Type I collagen albumen and fish-skin albumen processing tissue culture plate
(P<0.05)。
4 embedding cell scratch experiment result of table
* P < 0.05 compared with big rat-tail Type I collagen albumen of the invention
3 conclusions
It is compared with ox bone Type I collagen albumen and fish-skin albumen, the present invention is big, and rat-tail Type I collagen albumen is more advantageous to fell
The fibroblastic migration of skin, it was demonstrated that the big rat-tail Type I collagen albumen of the present invention is conducive to the reparation of skin.
Two, the skin-whitening functional verification experiment of big rat-tail Type I collagen albumen:
1 materials and methods
1.1 key instrument equipment
Experiment key instrument equipment.(being shown in Table 5)
The experiment key instrument equipment list of table 5
1.2 main agents
Main agents are shown in Table 6.
Table 6 predominantly detects reagent list
1.3 experimental method
1.3.1 mouse melanin tumor cell culture and processing
The culture of mouse melanin tumor cell cell culture according to provided by National Laboratory cellular resources shared platform refers to
Southing row is incubated at 37 DEG C, 5%CO with the DMEM-H culture medium containing 10%FBS2Cell incubator in, it is close to attached cell
When degree reaches 90% or so, with 0.05%Trypsin-EDTA vitellophag, after 500g centrifugation, it is resuspended with complete medium thin
Born of the same parents, 1:3 are passed on, and are passed on 1 time within 3 days.
After logarithmic growth phase cell dissociation, cell is counted, by 1 × 103、1×104、1×105A cell difference
96 orifice plates, 24 orifice plates, 6 orifice plates are inoculated in, after normal incubation medium culture for 24 hours, are added with the reagent for being diluted to exposed final concentration
Enter cultivating system, the final mass concentration of big rat-tail Type I collagen albumen, ox bone Type I collagen albumen and fish-skin albumen is respectively
The each concentration of 8mg/mL, 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL sets 4 holes, and rat is not added in control group
Tail Type I collagen albumen is incubated for 72h.Carry out relevant experimental implementation.
1.3.2 cell proliferation inhibition rate
Inoculated and cultured bottle is inoculated in 96 porocyte culture plates simultaneously, by rest part, and 7 groups, every group of 5 holes, every 100 μ L of hole,
37 DEG C of incubator stationary cultures；After culture for 24 hours, addition is prepared with 0.5%MTT (5mg/mL) from phosphate buffer to every hole
20uL, 37 DEG C of incubators are interior to be cultivated；Culture is terminated after 4h, and culture solution in hole is carefully sucked out.It is sub- that 150 μ L dimethyl are added in every hole
Sulfone sets low-speed oscillation 10min on shaking table, dissolves crystal sufficiently.Simultaneously every the first hole of row be set as zeroing hole (culture medium,
MTT, dimethyl sulfoxide).With each hole light absorption value of measurement at microplate reader OD570nm；Melanocyte proliferation inhibition rate=(1 one is each
Concentration mean absorbance values ÷ control group mean absorbance values) × 100%.
1.3.3 tyrosinase vitality test:
The final mass concentration of big rat-tail Type I collagen albumen, ox bone Type I collagen albumen and fish-skin albumen be respectively 8mg/
The each concentration of mL, 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL sets 4 holes, and big rat-tail I is not added in control group
Collagen type is incubated for 72h.Liquid is discarded supernatant, is rinsed twice with pH6.8PBS, every hole adds 90 μ L containing 1% (volume fraction)
The PBS of TritonX-100.Ultrasonication in ice bath, every hole add 10 μ L 1 × 10-2The L-dopa of moL/L, 37 DEG C of incubation 60min,
With 490nm wavelength colorimetric, each hole absorbance value is surveyed in blank well zeroing.Inhibitory activity against tyrosinase=(1 one each concentration are average
Absorbance value ÷ control group mean absorbance values) × 100%.
1.3.4 melanin content measures:
Mouse melanin tumor cell is inoculated in 36 orifice plates with the density of 1 × 105/mL.It is incubated in carbon dioxide incubator
After educating 24 hours, big rat-tail Type I collagen albumen, ox bone Type I collagen albumen and fish-skin albumen culture solution, concentration gradient is consistent,
Concentration is respectively 8mg/mL, 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, is contaminated 3 days, and supernatant is abandoned,
The digestion of 0.5mL pancreatin, constant volume then cell count is added in PBS washing, every hole.Remaining cell suspension 1500r/min centrifugation
10mln abandons supernatant, and 200 microlitres of distilled water are added and volume ratio is the ethyl alcohol of 1:1, (removing is some opaque for ether mixed liquor
Substance), it shakes up, is placed at room temperature for 15 minutes, 3000r/min is centrifuged 5 minutes.Supernatant is abandoned, 1mL is added in precipitating and contains 10%
The 1mol/L NaOH aqueous solution of (mass fraction) DMSO.80 DEG C of heating 30min (tool plug), select 490 nano wave lengths enzyme-linked
Absorbance value is surveyed in immune detector.Melanocyte growth rate=(drug hole cell density ÷ control wells cell density) ×
100%, B16 cell inhibiting rate=[1 one (drug hole absorbance value ÷ drug hole cell density) ÷ (control wells absorbance values
÷ control wells cell density)] × 100%.
2 results:
2.1 mouse melanin tumor cells proliferation
Big rat-tail Type I collagen albumen, ox bone Type I collagen albumen, each dosage group of fish-skin albumen are to mouse melanin tumor cell
Proliferation has no depression effect.It is shown in Table 7.
The measurement of 7 mouse melanin tumor cell proliferation inhibition rate of table
2.4 big rat-tail Type I collagen albumen, ox bone Type I collagen albumen, influence of the fish-skin albumen to tyrosinase activity:
Big rat-tail Type I collagen albumen, ox bone Type I collagen albumen, each dosage group of fish-skin albumen are to mouse melanin tumor cell
Tyrosinase activity significantly inhibits, and dose-response relationship is presented.When dosage is more than or equal to 0.5 mg/mL,
In isodose level, ox bone Type I collagen albumen and fish-skin albumen are significantly lower than big I type of rat-tail to TYR enzyme maximum inhibition
Collagen.It is shown in Table 8.
The influence of 8 tyrosinase activity of table
* the P < 0.05 compared with this product under concentration is indicated
2.5 big rat-tail Type I collagen albumen, ox bone Type I collagen albumen, fish-skin albumen embed the influence to B16 cell
Big rat-tail Type I collagen albumen, ox bone Type I collagen albumen, each dosage group of fish-skin albumen are to mouse melanin tumor cell
Melanin content significantly inhibits, and dose-response relationship is presented.When dosage is more than or equal to 0.5mg/ml, same
Equal dosage levels, ox bone Type I collagen albumen and fish-skin albumen are significantly lower than big rat-tail Type I collagen to TYR enzyme maximum inhibition
Albumen.It is shown in Table 9.
Influence of the big rat-tail Type I collagen albumen of table 9 to melanin
* the P < 0.05 compared with this product under concentration is indicated
Conclusion: the big rat-tail Type I collagen albumen of the present invention, ox bone Type I collagen albumen compare whitening effect more with fish-skin albumen
It is good.
Skin, eyes, the Genetic toxicity of embodiment 2, big rat-tail Type I collagen albumen
One, acute skin irritation is tested
It is tested as follows according to " cosmetics health specification " (version in 2007):
Material and method:
1, tested material: big rat-tail Type I collagen albumen (with formula in the present embodiment 1 and method preparation)
2, animal and feeding environment: New Zealand kind rabbit, regular grade, by, totally 4, weight 2-3kg.Beijing Jin Muyang
Experimental animal cultivates Co., Ltd [credit number: SCXK (capital) 2015-0005].Animal feeding place: Chinese disease control
Central animals room (credit number: SYXK (capital) 2014-0043) processed；The temperature of animal house is 20-25 DEG C, humidity 40-50%
RH.Animal feed is purchased from Beijing HFK Bio-Technology Co., Ltd. (credit number: SCXK (capital) 2014-0008), drink
Water is pure water.
3, test method: before test about for 24 hours, back part of animal backbone diamond wool being cut, the left and right each about 3cm of unhairing range
×3cm.The big rat-tail Type I collagen albumen of tested material is taken respectively about 0.5 to be coated directly on 2.5cm × 2.5cm skin, then with two layers of yarn
Cloth and one layer of glassine paper covering, then fixed with nonirritant adhesive plaster and bandage.Other side skin is as control.The application time
For h.Then with clear Xian to remove residual tested material.1h after removing tested material, for 24 hours, 48h, 72h and observe for the 4th, 5,6
The dermoreaction at position is smeared, " cosmetics health specification " (version in 2007) " skin irritation/corrosion test " Plays are pressed
Dermoreaction scoring is carried out, according to 24,48 and 72 hours each observation time point top mean values, is pierced by wherein standard determination skin
Swash intensity.
Test result: as shown in table 10.
The big rat-tail Type I collagen albumen of table 10 is to Rabbits with Acute skin irritation test result
By result in table 10 it is found that tested material is nonirritant to Rabbits with Acute skin irritation.
Two, acute ocular irritation test
It is tested as follows according to " cosmetics health specification " (version in 2007):
Material and method:
1, tested material: big rat-tail Type I collagen albumen.
2, animal and feeding environment: new zealand rabbit, regular grade, Beijing Jin Muyang experimental animal cultivate Co., Ltd
[credit number: SCXK (capital) 2015-0005], totally 3.Weight is 2-3kg.Animal feeding place: Chinese Center for Disease Control
Animal house (credit number: SYXK (capital) 2014-0043)；The temperature of animal house is 20~25 DEG C, and humidity is 40~50%RH.It is dynamic
Object feed is purchased from Beijing HFK Bio-Technology Co., Ltd. (credit number: SCXK (capital) 2014-0008), and drinking water is
Pure water.
3, test method: rabbit two are checked for 24 hours before test, selected animal is anophthalmia irritation, cornea
Defect and conjunctival damage.Tested material about 0.1ml is respectively dropped into 3 rabbit conjunctiva of right eye capsules when test, makes upper and lower eyelid quilt
Dynamic closure 1s, until the 4s faster normal saline flushing 30s of enough, flow velocity.Left eye, which is equally handled, makees own control.In contamination
Afterwards 1h, for 24 hours, 48h, 72h and animal eyes were checked in the 4th, 7 day, it is " acute by " cosmetics health specification " (version in 2007)
Eye irritation/corrosion test " Plays carry out Eye irritation reaction scoring, carry out eye irritation reaction classification by its Plays.
Test result: as shown in table 11.
11 tested material of table rinses Rabbits with Acute eye irritation result 30s
By result in table 11 it is found that the big rat-tail Type I collagen albumen of tested material is to Rabbits with Acute eye irritation result
It is nonirritant.
Three, skin phototoxicity is tested
It is tested as follows according to " cosmetics health specification " (version in 2007):
Material and method:
1, tested material: big rat-tail Type I collagen albumen
2, positive substance: 0.005%8- methoxyl group psoralea corylifolia.
3, animal and feeding environment: Albino guinea pig, regular grade, Beijing Jin Muyang experimental animal cultivate Co., Ltd
[credit number: SCXK (capital) 2015-0005], totally 6.Weight is female 220-260g, male 230-280g.Animal feeding
Point: Chinese Center for Disease Control's animal house (credit number: SYXK (capital) 2014-0043)；The temperature of animal house is 20-25 DEG C,
Humidity is 40-50%RH.Animal feed is purchased from Beijing HFK Bio-Technology Co., Ltd.'s (credit number: SCXK (capital)
2014-0008), it drinks water for pure water.
4, test method: UV light source: U.S.'s GE Products, diameter 38mm, power 40W, wavelength 350-400nm's
Black light tube.UVB<0.1J/cm2。
18-24h before testing, goes to hair-fields in cavy backbone two sides for 4 pieces (numbers 1,2,3,4), every piece is removed gross area about
It is located at left side for 2cm × 2cm, 1,3,2,4 are located at right side.When experiment, animal is fixed on special plank, about by tested material
0.2g is coated on hair-fields 1,2, and 3,4 do not apply tested material.1,3 areas are covered with aluminium-foil paper after applying tested material 30min, adhesive tape is solid
Fixed, 2,4 areas are irradiated with UV light source.Dermoreaction is observed respectively at 1,24,48 and 72h after irradiation, according to " makeup
Product hygienic practice " " skin phototoxicity test " Plays determine every reaction of animals scoring in (version in 2007).
Luminous intensity average value: 2.57mW/cm2；Irradiation time is calculated as follows as 65min.
Experimental result: as shown in table 12.
12 tested material of table is to guinea pig skin Phototoxicity experiment result
Note: 1,2,3,4 for " cosmetics health specification " (2007) second part seven, skin phototoxicity test shown in
Trial zone.
13 positive substance of table is to guinea pig skin Phototoxicity experiment result
Note: 1,2,3,4 for " cosmetics health specification " (2007) second part seven, skin phototoxicity test shown in
Trial zone.Positive controls date of test is 2 months 2018 on March 10th, 1 day 1.
By above-mentioned experimental result it is found that the big rat-tail Type I collagen albumen of the tested material is to guinea pig skin Phototoxicity experiment result
For feminine gender.
Four, Skin allergy test
Material and method:
1, cell strain: Chinese hamster ovary (CHO) cell strain.
2, metabolism activation system: using through sodium phenobarbital and β-naphthoflavene combined induction rat liver homogenate and plus accordingly
The mixture that is configured to of confactor as Metabolic Activation of Cyclophosphamide (S9mix)。
3, tested material: big rat-tail Type I collagen albumen is used to test after using the DMEM culture solution dilution without serum.
4, test method:
(1) culture solution: 10% fetal calf serum and the penicillin and streptomycin of 100IU/ml is added in DMEM culture solution.
(2) highest final concentration determines: test sets tested material group and negative control group.It is 6cm that Chinese hamster ovary celI, which is inoculated in diameter,
Culture dish in, inoculum density be 1.2 × 106/ ware, 37 DEG C, 5%CO2After culture for 24 hours, culture solution in culture dish is sucked, is added
S is added simultaneously in the tested material of various concentration and culture solution without serum, metabolism activation group9Mix is put and acts on 3h in incubator
Afterwards, the culture solution containing tested material is discarded, is washed cell 3 times with D-hanks liquid, the culture solution for containing 10% fetal calf serum is added, is continued
For 24 hours, negative control group only adds culture solution (metabolism activation group while S to be added for culture9).It is thin that each plate is observed under inverted microscope
The growing state of born of the same parents, the highest final concentration of tested material when determining test according to the change of cell level of coverage.
(3) chromosomal aberration test: test is carried out in the case where metabolism activation system condition is not added in adduction.If tested material group, yin
Property control group and positive controls.According to trial test as a result, the final concentration of tested material group is set as 250,125 and 62.5 μ g/mL
(-S9) and 2000,1000 and 500 μ g/mL (+S9).It is separately added into tested material according to processing method identical with trial test, is acted on
Liquid is changed after 3h, is continued culture and is harvested cell for 24 hours.Colchicine, final concentration of 1.0 μ g/mL is added in 4h before harvesting.According to a conventional method
Digestion, hypotonic, fixed, film-making, Giemsa dyeing.100 points are chosen to each tested material group, negative control group and positive controls
Good division phases cell is dissipated, chromosome aberration analysis is carried out.The type and number of chromosomal structural aberration are recorded, and is counted
Calculate chromosome aberrations rate.
Statistical analysis: χ is used2Inspection compares the chromosome aberrations rate of each tested material group cell with negative control group
Compared with, it is determined whether there is significant difference.
Test result:
14 mammalian cells in vitro chromosomal aberration test result (- S of table9)
*P<0.01
15 mammalian cells in vitro chromosomal aberration test result (+S of table9)
*P<0.01
By above-mentioned experimental result it is found that under this experiment condition, the sample is in the mammalian Chinese storehouse in vitro culture
In mouse gonad cell (CHO) chromosome aberration detection system, compared with negative control group, regardless of whether activation system is added, greatly
Rat-tail Type I collagen albumen does not cause obviously increasing for chromosome aberrations rate, therefore the not shown mutagenicity in this test macro.
Embodiment 3, big rat-tail Type I collagen albumen toxicology test
1 material and method
1.1 samples: big rat-tail Type I collagen albumen is colorless and transparent g., jelly-like colloid.
1.2 experimental animals and rearing conditions: animal: Beijing HFK Bio-Technology Co., Ltd. is selected to provide
SD rat (cleaning grade, quality certification number: SYXK (capital) 2014-0004).Raise place: Chinese Center for Disease Control's animal house is (perhaps
It can the number of card: SYXK (capital) 2014-0043)；Feed: purchased from Beijing HFK Bio-Technology Co., Ltd. (credit number:
SCXK (capital) 2014-0004).
1.3 key instruments and reagent: BECKMAN GS15R centrifuge (608112)；7080 type automatic biochemical analyzer of Hitachi
(060817)；BECKMAN COULTER Ac.T diff2TMBlood analyser (060816)；NIKON biomicroscope
(608105)；VIP-E150F fully-automatic dewatering machine (630125), the full-automatic embedding machine of TEK-CC/TEK-EC (630122),
The semi-automatic paraffin slicing machine of E0106 (630121), ST5010 full-automatic dyeing machine (630127), the full-automatic mounting of SCA-5600
Machine (630126).Kurt balanced electrolyte solution and Kurt hemolytic agent are limited purchased from Beckman Kurt experimental system (Suzhou)
Company；Blood biochemistry detection kit is purchased from Zhongsheng Beikong Biological Science & Technology Co., Ltd..
1.4 experimental methods:
1.4.1 it rat acute toxicity test: is carried out using maximal tolerance dose method (MTD).Select healthy SD rat 20
(each 10 of male and female) are tested.Rat body weight is 180-210 grams.With distilling, water-soluble to 180ml, (concentration is 45g tested material
0.25g/ml), stomach-filling is carried out with 20ml/kgBW stomach-filling amount, 3 times a day, i.e., acute toxicity dosage is 15g/kg BW, after stomach-filling
It is observed continuously 14 days.Record animal poisoning performance and death condition.
1.4.2 genetic toxicity test:
1.4.2.1 Salmonella reversion test: using identified satisfactory salmonella typhimurium histidine deficient TA97,
Tetra- plants of test strains of TA98, TA100, TA102 are tested.It is made of the rat liver homogenate of Polychlorinated biphenyls (PCB) induction
S-9 is as activation system.According to toxicity test as a result, test sets 0.313,0.625,1.250,2.500,5,5.000mg/ ware
1g tested material is used after disinfection by ultraviolet light and is settled to 20ml as maximum dose level using sterile water dissolution (5.0mg/ ware is dense by dosage
Degree), for remaining dosage with 2 times of doubling dilutions of sterile water, sample-adding amount is 100 μ L/ wares.Untreated control, solvent control are set simultaneously
With positive control ware.It is mixed that 0.1ml test strain enrichment liquid, 0.1ml tested material solution and 0.5ml S-9 are added in top agar
It closes liquid (when needing metabolism activation), is poured into after mixing on bottom culture medium flat plate.In 37 DEG C of culture 48h, counts every ware and return change
Clump count.If tested material return become clump count be solvent control clump count 2 times or more, and have dose-response relationship person if
It is set to the positive.Entire test repeats to do under the same conditions primary.
1.4.2.2 mice bone marrow micronucleus: using interval, oral administration by gavage is tried twice for 24 hours
It tests.25-30 grams of mouse of weight 50 is selected, is randomly divided into 5 groups by weight, every group 10, half male and half female.Weigh 20g tested material
Use distilled water to be dissolved to 80ml as high dose, in, successively 2 times of doubling dilutions, stomach-filling amount are 30ml/kgBW to low dosage, i.e.,
The big basic, normal, high dosage of rat-tail Type I collagen albumen is respectively 1.88,3.75,7.50g/kgBW.With the ring phosphorus of 40mg/kgBW dosage
Amide is positive control, and distilled water is negative control.Last gives cervical dislocation after big rat-tail Type I collagen albumen 6h to put to death animal,
Bone marrow of sternum calf serum is taken to dilute smear, methanol is fixed, Giemsa dyeing.Under biology microscope, every zoometer
Polychromatic erythrocyte (PCE) number in 200 red blood cells (RBC) of number, and calculate its proportion；Every animal counts 1000
A polychromatic erythrocyte, microkernel incidence carry out statistical disposition in terms of the PCE permillage containing micronucleus.
1.4.3 it feeding test for 90 days: selects weight 60-90g to wean rat 80, is randomly divided into 4 groups, i.e. control group and 3
A tested material group.Weighing 20g tested material uses distilled water to be dissolved to 80ml as high dose (being maximum using concentration), in, low dose
Successively 2 times of doubling dilutions, stomach-filling amount are 10ml/kgBW to amount.I.e. high, medium and low dosage is respectively 0.63,1.25,2.50g/kg
Distilled water is given in BW, control group stomach-filling.Every group of 20 animals, half male and half female.Animal single cage is fed, and free diet weighs weekly
Rat body weight twice adjusts stomach-filling amount according to weight.It is recorded twice weekly to appetite, surplus food and berley amount, is observed continuously 90 days,
Experiment terminates blood sampling and surveys every hematology, blood biochemistry index and histopathologic examination.
1.4.3.1 observation index:
1.4.3.1.1 the general performance of animal, weight, food utilization
1.4.3.1.2 blood routine and biochemical indicator: white blood cell count(WBC) and its classification, red blood cell count(RBC), hemoglobin, blood are small
Plate counts；Glutamic-oxalacetic transaminease, glutamic-pyruvic transaminase, alkaline phosphatase, urea nitrogen, creatinine, cholesterol, triglycerides, blood glucose, total egg
White, albumin.
1.4.3.1.3 dirty body ratio: experiment, which terminates to put to death animal, takes liver,kidney,spleen, heart, thymus gland and testis weighing, calculates phase
The dirty body ratio answered (with the calculating of hectogram weight).
1.4.3.1.4 Histopathology: dissection carries out gross examination of skeletal muscle and histopathologic examination.To each dosage group animal
When making gross examination and not finding that obvious lesion and biochemical indicator no abnormality seen change, only first to high dose group and control animals into
Row heart, adrenal gland, thymus gland, liver, kidney, stomach and duodenum, spleen, testis or ovary pathological observation.
1.4.3.2 statistical method: data calculate mean and standard deviation through SPSS10.O, and for statistical analysis.
2 results
2.1 rat acute toxicity tests: by table 1,6 as it can be seen that female, male rat 15.0g/kgBW dosage is given in oral stomach-filling
Big rat-tail Type I collagen albumen, observation 14 days after, apparent poisoning symptom is had no, also without death, the results showed that the tested material pair
Female, male rat MTD is greater than 15.0g/kgBW.
The big rat-tail Type I collagen albumen rat acute toxicity test result of table 16
| Gender | Initial weight (g) | Weight (g) eventually | MTD(g/kgBW) |
| It is female | 194.6±5.8(185-206) | 230.2±10.1(215-246) | >15 |
| It is male | 196.2±6.8(182-210) | 294.6±17.0(259-319) | >15 |
2.2 genetic toxicity tests:
2.2.1 Salmonella reversion test: by table 17 and table 18 as it can be seen that control ware, which returns, becomes clump count in normal range (NR), big I type of rat-tail
Each dosage group of collagen returns change clump count and is less than 2 times of solvent control clump count, also without dose-response relationship, therefore to mouse
Tetra- plants of test strains of salmonella typhi TA97, TA98, TA100, TA102 are showed no big I type of rat-tail when adding and S-9 being not added
Collagen, which has, causes gene mutation effect.
Table 17 is rat-tail Type I collagen albumin A mes big to test the 1st result
Note: result above is three ware average values
Table 18 is rat-tail Type I collagen albumin A mes big to test the 2nd result
Note: result above is three ware average values.
2.2.2 mice bone marrow micronucleus: by table 19 as it can be seen that each thermophilic polychromatophilia of tested material dosage group is red thin
Born of the same parents (PCE) percentage is no less than the 20% of negative control, shows tested material no cytotoxicity under test dose；No matter male is gone back
It is female mice cyclophosphamide positive controls microkernel incidence obviously higher than negative control group and each dosage group of tested material (pool
Loose distribution inspection P<0.01), and each dosage group of tested material there are no significant compared with negative control group difference (P>0.05).It says
The bright tested material is to Mouse Somatic Cells chromosome without mutagenesis.
Influence of the big rat-tail Type I collagen albumen of table 19 to Micronuclei In The Mouse Bone Marrow incidence
Note:a: show compared with each tested material processing group, Poisson distribution counts P > 0.05；
b: show compared with each tested material processing group and negative control group, Poisson distribution counts P < 0.01.
2.3 feeding test for 90 days:
2.3.1 upgrowth situation and food utilization:
Groups of animals activity, growth are normal, and coat is dense glossy.By table 20 as it can be seen that with 0.63,1.25,2.50g/
The big rat-tail Type I collagen albumen stomach-filling of kgBW dosage gives rat 90 days, each tested material dosage group the weight of animals, weight gain
With total food utilization rate there are no significant compared with the control group difference (P > 0.05).
Influence of the big rat-tail Type I collagen albumen feeding test for 90 days of table 20 to rat body weight
2.3.2 blood routine and blood biochemistry index:
By table 21-22 as it can be seen that with 0.63,1.25, the big rat-tail Type I collagen albumen stomach-filling of 2.50g/kgBW dosage gives greatly
Mouse 90 days, compared with the control group, the blood routine of each tested material dosage group of male and female there are no significant compared with the control group difference (P >
0.05)。
Influence of the big rat-tail Type I collagen albumen feeding test for 90 days of table 21 to rat mid-term blood routine
Influence of the big rat-tail Type I collagen albumen feeding test for 90 days of table 22 to rat latter stage blood routine
By table 23-24 as it can be seen that with 0.63,1.25, the big rat-tail Type I collagen albumen stomach-filling of 2.50g/kgBW dosage gives greatly
Mouse 90 days, compared with the control group the blood biochemistry index of each tested material dosage group there are no significant compared with the control group difference (P >
0.05)。
Influence of the big rat-tail Type I collagen albumen feeding test for 90 days of table 23 to rat mid-term blood biochemistry index
Influence of the big rat-tail Type I collagen albumen feeding test for 90 days of table 24 to rat latter stage blood biochemistry index
2.3.3 general pathology and histological examination: by table 25 as it can be seen that with 0.63,1.25, the rat of 2.50g/kgBW dosage
Tail Type I collagen albumen stomach-filling gives rat 90 days, each tested material dosage group slaughter weight, liver, kidney, spleen and testis weight
Amount and the dirty body of each internal organs are than there are no significant compared with the control group difference (P > 0.05).
Influence of the big rat-tail Type I collagen albumen feeding test for 90 days of table 25 to Rats Organs and Tissues weight and dirty body ratio
By above-mentioned table 25 it is found that gross anatomy, which visually observes, does not find that each internal organs are abnormal.Rats'liver, kidney, stomach and 12
Duodenum 12, spleen, testis (or ovary) internal organs histological indications are as follows: liver: normal configuration can clearly debate knowledge, lobuli hepatis arrangement
Neatly, liver cell is in substantially radial trend by axle center of central vein；Most of liver cell form is normal, and part of hepatocytes can
Degree of taking a favourable turn hydropic degeneration；1 jenny (1/20) of 1 female animal (1/20) of control group and 2.50g/kgBW dosage group can
See that the liver cell focal necrosis lesion is often occurred in experimental animal.Kidney: structure can clearly identify that the nephron is in uniform point
Cloth, glomerulus and renal tubule structure are normal, have no the lesions such as interstitial cell hyperplasia, inflammatory cell exudation and glomerulus atrophy；Portion
Divide renal tubules,convoluted epithelial cell hydropic degeneration.Heart: institutional framework can identify, be made of myocardium and the internal membrane of heart, the external membrane of heart.The heart
Muscle fibre is the striated muscle cell of multicore, branch, there is a small amount of connective tissue and blood vessel therebetween in being staggered.Spleen: spleen tissue
Structure is easy to recognize, and the little beam-like interval for forming connective tissue is stretched from envelope to deep layer, and blood vessel is walked wherein, white pulp and red pulp
Normally, accidental snius lienis slightly expands extravasated blood.Above-mentioned liver, kidney, heart and spleen change into the common lesion of experimental animal, right
According to group and high dose group without group difference, therefore think unrelated with tested material.Stomach: stomach wall structure is readily identified, mucous epithelium form
Normally, bleeding, necrosis and inflammatory exudation, the variation also without gland cell composition are had no.Duodenum: mucous membrane, submucosa, muscle layer
And each layer of serous coat can be recognized clearly, each layer structure and its cell are normal, and mucous membrane has no bleeding, necrosis and inflammatory cell infiltration.
Testis: each component part is readily identified, and convoluted seminiferous tubule limitans is clear, it is seen that first spermatocyte, secondary spermatocyte, sperm are thin
Born of the same parents and sperm have no that bleeding, necrosis and germ cell development are abnormal.Ovary: structure composition is readily identified, visible different hairs in cortex
The ovarian follicle (primordial follicle, primary follicle, secondary follicle and graaffian follicle) and corpus luteum for educating the stage, have no ovarian follicle dysplasia and
Organize the pathological changes such as internal haemorrhage, inflammation.Thymus gland: surface is by with thin layer connective tissue coating, and the visible thymic tissue in lower section is just
Normal structure divides cortex and medullary substance two parts.Between occasionally thering is a small amount of fat cell to mix thymic tissue.Have no obvious pathological change.It is right
According to group and experimental group without significant difference.Adrenal gland: surface is by with thin layer connective tissue coating, and the visible adrenal tissue in lower section is just
Normal structure divides the cortex and central portion medullary substance two parts on surface layer.Have no obvious pathological change.Liver,kidney,spleen, Stomach duodenum,
Testis or ovary, heart, thymus gland and adrenal tissue are showed no the pathological change of meaning.
3 brief summaries:
3.1 acute toxicity tests: to the oral acute toxicity of female, male rat, MTD is all larger than big rat-tail Type I collagen albumen
15.0g/kgBW。
3.2 genetic toxicity tests: Salmonella reversion test, mice bone marrow micronucleus are showed no I type glue of rat-tail
Former albumen has mutagenesis.
3.3 feeding test for 90 days: with 0.63,1.25, the rat-tail Type I collagen albumen stomach-filling of 2.50g/kgBW dosage gives
Rat 90 days, as the result is shown: each tested material dosage group animal activity, growth were normal, each tested material dosage group the weight of animals, weight gain
With food utilization there are no significant compared with the control group difference (P > 0.05).Each dosage group of peripheral blood cell counts index and control group
Compare, the routine blood indexes of each tested material dosage group of male and female there are no significant compared with the control group difference (P > 0.05)；Blood biochemistry
Index compared with the control group, the blood biochemistry index of each tested material dosage group of male and female there are no significant difference (P > 0.05)；It is each tested
Object dosage group heart, thymus gland, liver, kidney, the weight of spleen and testis and the dirty body of each internal organs are than compared with the control group without significantly
Sex differernce (P > 0.05)；Pathological index also has no the abnormal change as caused by tested material.Therefore have no tested material to the weight of animals, food
There are adverse effect in object utilization rate, hematology, blood biochemistry, dirty body ratio and histopathologic examination.
Claims (5)
1. application of the big rat-tail Type I collagen albumen in the product for preparing wrinkle resistant moisturizing and/or whitening.
2. application according to claim 1, it is characterised in that: the product includes in food, cosmetics and health care product
It is at least one.
3. a kind of cosmetics, it is characterised in that: the cosmetics include that the component of following mass parts is made: big rat-tail Type I collagen egg
White 2~3 parts；0.5~1 part of peony extract；0.6~1.2 part of propolis extract；3 parts of Nexbase 2004；Caprylic capric three is sweet
4 parts of grease；3 parts of dimethicone；1.2 parts of cetanol；0.5 part of docosyl alcohol；4 parts of different tridecanol isononoate；100 parts of water；It is sweet
3 parts of oil；3 parts of butanediol；1 part of glycerol stearate (SE)；- 20 1.5 parts of docosyl alcohol polyethers；0.03 part of disodium ethylene diamine tetraacetate；
0.5 part of copolymer of sodium acrylate acyl dimethyltaurine sodium；0.2 part of preservative；0.1~0.3 part of essence.
4. 1) preparation method of cosmetics in claim 3 includes the following steps: Nexbase 2004, caprylic capric triglycerin
Ester, dimethicone, cetanol, docosyl alcohol and the mixing of different tridecanol isononoate, heating obtain A phase；
2) by big rat-tail Type I collagen albumen, peony extract water, glycerol, butanediol, glycerol stearate, docosyl alcohol polyethers -20
It is mixed to get B phase with disodium ethylene diamine tetraacetate, then the A obtained in step 1) is added in the B phase, pre- cream
Change, homogeneous；
3) through A phase described in step 2) with the B phase after processing, it is mixed that sodium acrylate acyl dimethyltaurine sodium copolymer is added
It closes；
4) step 3) treated system is cooling, preservative, essence homogeneous is added then to get the cosmetics are arrived.
5. according to the preparation method in claim 4, it is characterised in that: described to be heated to 80 DEG C in step 1)；
In step 2), the pre-emulsified time is 5min, and the time of the homogeneous is 3min；
In step 3), the system is cooled to 45 DEG C；
In step 4), the time of the homogeneous is 2min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008032054A2 (en) * | 2006-09-15 | 2008-03-20 | Novathera Limited | Composite material comprising a tissue matrix compound and a bioactive glass |
| CN109431850A (en) * | 2018-03-16 | 2019-03-08 | 广东众尔健生物科技有限公司 | A kind of application of Collagen type-I in cosmetic field |
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2019
- 2019-06-17 CN CN201910520678.5A patent/CN110200839A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008032054A2 (en) * | 2006-09-15 | 2008-03-20 | Novathera Limited | Composite material comprising a tissue matrix compound and a bioactive glass |
| CN109431850A (en) * | 2018-03-16 | 2019-03-08 | 广东众尔健生物科技有限公司 | A kind of application of Collagen type-I in cosmetic field |
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| Title |
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| 冯年平等: "《中药经皮给药与功效性化妆品》", 31 May 2019, 中国医药科技出版社 * |
| 宝拉•培冈等: "《美丽圣经：升级版》", 30 April 2017, 广西师范大学出版社 * |
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