CN110339147A - A kind of composition for external application and application thereof - Google Patents
A kind of composition for external application and application thereof Download PDFInfo
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- CN110339147A CN110339147A CN201811429725.7A CN201811429725A CN110339147A CN 110339147 A CN110339147 A CN 110339147A CN 201811429725 A CN201811429725 A CN 201811429725A CN 110339147 A CN110339147 A CN 110339147A
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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
- A61K8/55—Phosphorus compounds
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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
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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
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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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
- A61Q19/08—Anti-ageing preparations
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Abstract
The invention belongs to skin repair fields, provide a kind of composition for external application and application thereof, composition for external application of the present invention includes mescenchymal stem cell excretion body freeze-dried powder, wherein, at least contain 25.0~35.0ng/g of fibronectin in the freeze-dried powder, 5.0~14.5ng/g of vascular endothelial growth factor, 5.5~15.5ng/g of fibrous bud Porcine HGF, 5.0~10.0ng/g of epithelial cell growth factor, 25.0~30.0ng/g of hepatocyte growth factor, 7.5~17.5ng/g of transforming growth factor, 0.5~5ng/g of sphingosine 1-phosphate, 0.5~5ng/g of ceramide -1- phosphoric acid.Composition for external application provided by the invention contains to skin mescenchymal stem cell excretion body freeze-dried powder having repairing effect, without containing medium component and cellular component, it avoids addition and has injury of the cytotoxic substance to human body and skin, avoid the potential hazard that allergy occurs for skin.Product property is more stable after being prepared into freeze-dried powder, more muchly to play its bioactivity.
Description
Technical field
The invention belongs to skin repair technical fields, and in particular to a kind of composition for external application and application thereof.
Background technique
Skin is the maximum organ of human body, is covered in human body surface, have the function of protection, feel, adjust body temperature etc., structure
It makes from top to bottom, is followed successively by epidermis (epidermis), skin corium (dermis) and subcutaneous tissue (hypodermis).Generally
The reason of with the growth at age, the characterization of skin aging can occur people gradually, skin aging can be divided into endogenous and exogen.
The aging of endogenous refers to the natural ageing process of body, and including the growth with the age, cutin stem cell can be by
It is decrescence few, cause basal layer cell disorder, sodium hyaluronate reduction and moisturizing factor to be lost；Point of hormone (such as sebaceous glands)
The metabolism of whole skin can be slowed down by secreting decline, and skin is finally made to lack tension and elasticity；The fibroblast of skin corium
(fibroblast) hypofunction, and manufacture collagen and elastin laminin are tapered off, so that the connective tissue of skin corium
Into catagen phase, causes cutis laxa, lacks lines, or even generate wrinkle；The degeneration of skin corium connective tissue will affect skin
The water storage function of skin leads to dry skin and water shortage；And the gradually atrophy of epidermis and skin corium, cause under epidermis phylactic power defensive power
Wound is dropped and be easy to produce, or blood vessel elasticity is caused to be deteriorated, be easy to cause subcutaneous hemorrhage.
The aging of exogen is then directly contacted with external environment because of skin, by external factor (such as solarization, pollution, from
Injured and smoked as base) stimulation and caused by aging phenomenon, update including cell and reparation be suppressed, the protection of skin
Function is deteriorated and melanin deposition.Wherein, the primary factor for causing skin aging is to be exposed to sunlight caused ultraviolet light
Damage, daylight can accelerate 5 to 10 times of aging speed of skin.Ultraviolet light, predominantly long wave in contact with general daily life
Ultraviolet B radiation (UVB) in ultraviolet light and ultraviolet light contacts ultraviolet B radiation for a long time, and the fiber of skin corium can be stimulated female thin
Born of the same parents generate Collagenase (collagenase) and decompose collagen, and collagen density reduction and elastin laminin is caused to become
Property, so that skin is followed the string；The functional deterioration of melanocyte and cause the colour of skin uneven；And skin generates oneself excessive
Cause the age spot of sunburn and out-of-flatness to be formed by base, or even causes various cutaneum carcinomas.
Currently, there are many methods prevented and repair skin aging, including extracellular supported matrix replacement therapy, injection meat
Bacillus venenosus and the treatment of physical exfoliative.Wherein, extracellular supported matrix replacement therapy is included in skin surface wiping collagen
Albumen, elastin, mucopolysaccharide, sodium hyaluronate, vitamin e etc., however because the structure of these molecules is too big, therefore can not be effective by skin
It absorbs, hold time typically not greater than nine months into skin corium, and in vivo, therefore therapeutic effect is limited.Injection clostridium botulinum
Though the aging phenomenon of cutis laxa can be improved, the effect be usually only capable of maintain 3 to 6 months, and be easy to cause upper eyelid it is sagging,
The side effects such as expression is stiff strange or muscle of injection site is powerless.It is treated as physical exfoliative, though it can remove old
Change cutin, stimulate neoplastic skin hyperplasia, but is easy to produce the side effects such as wound, skin recovery is slow, postoperative complications are more.It is related
Another treatment trend of skin aging is cell therapy, mainly improves damaged skin tissue using stem cell.
Stem cell (stem cells, SC) is that a kind of pluripotency with the of self-replication capacity (self-renewing) is thin
Born of the same parents, under certain condition, it can be divided into multiple functions cell.Stem cell is a kind of inabundant differentiation, and still immature is thin
Born of the same parents, have the potential function for regenerating various histoorgans and human body, and medical field is referred to as " general-purpose cell ".Functionally, it does
Cell is defined as the of self-replication capacity and generates the cell of noble cells ability.More precisely, stem cell can produce
Raw progeny cell (self-replacation) identical with its parental cell, and the limited progeny cell of differentiation potential can be generated and (broken up thin
Born of the same parents).Stem cell can in body life process indefinite duration divide, and in suitable condition or after giving appropriate condition, energy
Differentiation generates a variety of different cell types.The reduction of human epidermal stem cell, power of regeneration reduce, and the death of epidermal cell is de-
It falls, hypofunction etc., as the skin spots that accumulate over a long period, wrinkle such as increase at the sign of agings.
The excretion body of source of human stem cell is that stem cell secretes under the stimulation such as tranquillization or oxygen deprivation stress, irradiation, oxidative damage
The extracellular vesica generated can serve as stem cell and differentiated thin by the transport protein matter of selectivity, mRNA, microRNA
The signaling molecule of born of the same parents.Existing research is proved mescenchymal stem cell excretion body and is reducing myocardial infarction area, mitigates limb ischemia,
Promote wound healing, improve graft versus host disease(GVH disease) (graft-versus-hostdisease, GVHD), reduce injury of kidney, promotees
Into liver regeneration, mitigates retinal damage and improvement cartilage and osteanagenesis etc. play a significant role.Excretion body is iuntercellular
The important carrier of information interchange, and excretion bulk properties is related with the cell in its source, thus the function of stem cell excretion body and its
The function of stem cell is closely related.Containing there are many growth factor and cell factor in excretion body, such as vascular endothelial growth factor is done
Cell factor etc. has important physiology application.
Then, but due to mescenchymal stem cell excretion body extract that there are recovery rates low, liquefied be not easy to save etc. it is all
More disadvantages cause to lack the skin care item containing mescenchymal stem cell excretion body in the prior art, the application in skin repair field
Still belong to blank.
Summary of the invention
In order to overcome the shortcomings in the prior art, the present invention provides a kind of new composition for external application and preparation method thereof
And purposes.
The present invention provides a kind of composition for external application, which is characterized in that is lyophilized comprising mescenchymal stem cell excretion body
Powder, wherein at least contain 25.0~35.0ng/g of fibronectin, blood vessel endothelium in the mescenchymal stem cell excretion body freeze-dried powder
5.0~14.5ng/g of Porcine HGF, 5.5~15.5ng/g of fibrous bud Porcine HGF, epithelial cell growth factor 5.0
~10.0 ng/g, 25.0~30.0ng/g of hepatocyte growth factor, 7.5~17.5ng/g of transforming growth factor, 1- phosphoric acid sheath ammonia
0.5~5ng/g of alcohol, 0.5~5ng/g of ceramide -1- phosphoric acid；Preferably, in the mescenchymal stem cell excretion body freeze-dried powder
At least contain fibronectin 30.0ng/g, vascular endothelial growth factor 12.0ng/g, fibrous bud Porcine HGF
12.5ng/g, epithelial cell growth factor 8.0ng/g, hepatocyte growth factor 26.0ng/g and transforming growth factor 1 2.5ng/
G, sphingosine 1-phosphate 4ng/g, ceramide -1- phosphoric acid 3ng/g.
Further, in above-mentioned composition for external application, the preparation method of the mescenchymal stem cell excretion body freeze-dried powder
It is as follows, comprising the following steps:
S1 is collected: being collected mescenchymal stem cell, is obtained mescenchymal stem cell single cell suspension；Preferably, it is filled between described
Matter stem cell includes that mesenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, fat mesenchymal are dry thin
Born of the same parents or muscle mesenchymal stem.
S2 culture: above-mentioned mescenchymal stem cell single cell suspension is inoculated into culture medium and is cultivated；
S3 is extracted: the above-mentioned culture medium containing mescenchymal stem cell being extracted and obtains the extraction of mescenchymal stem cell excretion body
Liquid；
S4 freeze-drying: by above-mentioned mescenchymal stem cell excretion body extracting solution be lyophilized to get.
Further, wherein cell culture processes described in step S2 the following steps are included:
A. will collect obtained mescenchymal stem cell single cell suspension be placed in 37 DEG C, volume fraction be 4%~8%CO2Training
It supports and is cultivated in case, cell fusion passes on after reaching 80%-95%, collects 3-5 for cell as culture cell；Preferably, described
CO2Volume fraction be 5~6.5%；Cell fusion is 90%；Cell is the 4th generation cell；
B. the cell for being in logarithmic growth phase in culture cell is chosen, serum free medium DMEM/F12, culture 48 is added
~96h collects supernatant daily, and replaces fresh culture medium, and the supernatant for collecting culture is used for the extraction of excretion body；It is excellent
Choosing, the incubation time is 72h.
Further, wherein extraction step described in step S3 specifically includes:
A. by the supernatant collected in step S2 at 4~6 DEG C, 20~30min is centrifuged with 10000~15000r/min,
Supernatant is stayed, precipitating is discarded；
B. by supernatant obtained by a step at 4~6 DEG C, 60~80min is centrifuged with 15000~30000r/min, stays supernatant
Liquid discards precipitating；
C. by supernatant obtained by b step at 4~6 DEG C, 60~80min is centrifuged with 10000~15000r/min, is discarded
Clear liquid retains precipitating to get mescenchymal stem cell excretion body extracting solution.
Further, wherein freeze drying process described in step S4 the following steps are included:
A. freeze drying protectant is added: freeze drying protectant is added in Xiang Shangshu mescenchymal stem cell excretion body extracting solution, and adjusts
PH value is 6.5；Contain following component: 0.3~0.5g of ascorbic acid, dextran 2.5 in the every 100ml of freeze drying protectant
~3.0g, trehalose 2g, 1~20g of antifreeze protein mixture, 10~20ml of the tert-butyl alcohol；Preferably, antifreeze protein mixing
Object is antifreeze glycoprotein (AFGP), I type antifreeze protein (AFP I) and II type antifreeze protein (AFP II) mixture, three's mass
Than for 2:1:1；
B. be lyophilized: freeze-drying pressure is 10~30pa, and temperature is -25~-30 DEG C, and the time is 10~12h.
Further, any of the above-described composition for external application, which is characterized in that also containing acceptable load in cosmetics
Body, including at least two or more in antioxidant, stabilizer, solubilizer, pigment, fragrance.
Further, the dosage form of the composition for external application is solution, lotion, gel, creams, lotion, pulvis, fertilizer
Soap, the detergent containing surfactant, any one in finish.
Further, the composition for external application be toner, facial mask essence, lotion, gel, creams, essential oil,
Cleanser, cleaning foam, clean water or spray.
Further, the composition for external application is toner, which is characterized in that mescenchymal stem cell excretion body extracts
Liquid secretes body freeze-dried powder 5.0~8.0%, glycerol 1%, polyalcohols A 3~5%, glycine betaine 0.5%, panthenol 0.8%, glycyrrhizic acid
Dipotassium 0.05~0.1%, other active components A 0.11~1.0%, polyethylene glycol -3 1.0~1.5%, surfactant A
0.25~0.3%, preservative A 0.01~0.25%, xanthan gum 0-0.15%, fragrance 0.03%, surplus are purified water；Wherein,
The polyalcohols A is one of butanediol, 1,2- pentanediol and dipropylene glycol or a variety of；The other active components A
For one of allantoin, niacinamide and Sodium Hyaluronate or a variety of；The surfactant A is hydroxyethyl cellulose, poly- mountain
One of pears alcohol ester -20 and Cremophor RH40 are a variety of；The preservative A is methyl hydroxybenzoate and/or Phenoxyethanol.
Further, the composition for external application be facial mask essence, the component comprising following weight percentage:
Mesenchymal stem cells excretion body freeze-dried powder 5.0~8.0%, glycerol 1%, polyalcohols B 3~5%, panthenol 0.8%, glycyrrhizic acid two
Potassium 0.05~0.1%, allantoin 0.2~0.3%, other active components B 0.5-2.0%, surfactant B 0.05-
3.6%, preservative B 0.25-0.45%, xanthan gum 0.2-0.4%, fragrance 0.05%, and aminomethyl propanol is added, the ammonia
The content of methylpropanol, which is subject to, adjusts the facial mask essence pH value to 5.5-7, and surplus is purified water；The polyalcohols
B is butanediol and/or 1,3- propylene glycol；The other active components B is niacinamide, Sodium Ascorbyl Phosphate, tocopherol second
One of acid esters and Sodium Hyaluronate are a variety of；The surfactant B be hydroxyethyl cellulose, isopropyl isostearate,
One of -2 oleate of polyglycereol and Natrulon H-10 oleate are a variety of；The preservative B is Nipasol, methyl hydroxybenzoate
Or one of Phenoxyethanol or a variety of.
Further, the composition for external application is lotion, and the component comprising following weight percentage: mesenchyma is dry
Cell excretion body freeze-dried powder 5.0~8.0%, glycerol 1%, 1,3-PD 3-5%, panthenol 1%, tocopherol acetate 0.5%,
Other active components C 0.1-2.0%, dimethyl silicone polymer 0.5-2%, emulsifier A165 0.5-2%, isostearic acid isopropyl
Ester 0.5%, oils C 0.2-3%, cetostearyl alcohol 0.8%, preservative C 0.1%, xanthan gum 0.5%, EDTA-2Na
0.1%, and triethanolamine is added, the content of the triethanolamine is subject to the pH value for adjusting the lotion to 5.5-7, and surplus is
Purified water；Wherein, the other active components C is one of dipotassium glycyrrhizinate, niacinamide and Parleam or a variety of；
The oils C is dormant oil and/or isohexadecane；The preservative C is methyl hydroxybenzoate and/or Nipasol.
The present invention also provides any of the above-described composition for external application to combine in the external preparation for skin that damage skin is repaired in preparation
Purposes in object.
The present invention also provides any of the above-described composition for external application to promote skin metabolism or anti-aging in preparation
Application in composition for external application.
Beneficial effect
Compared with prior art, the present invention having the advantage that
1. the mescenchymal stem cell excretion body of method preparation provided by the invention, does not contain medium component and groups of cells
Point, injury of the external additive to human body and skin is avoided, the potential hazard that allergy occurs for skin is avoided.
2. product property is more stable after purifying is prepared into freeze-dried powder, more muchly to play its bioactivity；And
It is higher using active cytokine content in the freeze-dried powder of this method preparation, for establishing skin barrier and repairing skin injury
Effect it is more preferable.
3. the freeze-dried powder of freeze drying protectant that the present invention uses preparation, be not added fetal calf serum commonly used in the art and
DMSO.A possibility that animal pathogenic pollutes stem cell is not only avoided, also avoids that there is cytotoxicity object using DMSO etc.
It verifies the irreversible damage caused by cell, for freezing with significant meaning for stem cell.
4. present invention employs contain following a few class key components in novel freeze drying protectant:
Trehalose (Trehalose) is by two glucose molecules with a, a, 1, and 1- glycosidic bond constitutes nonreducing sugar, from
Body property is highly stable, and trehalose has magical protective effect to organism, is because trehalose is in high temperature, high and cold, hypertonic
Unique protective film can be formed in cell surface under the severe environmental conditions such as pressure and dry dehydration thoroughly, is effectively protected protein point
Sub- invariance inactivation, thus the life process and biological characteristic of the body that sustains life.It is many to extraneous adverse circumstances, show outstanding
The species of degeneration-resistant tolerance, all there is a large amount of trehalose in vivo with them has direct relationship.With such as sucrose, Portugal in nature
Other carbohydrates such as grape sugar, do not have this function.This unique functional characteristic, so that trehalose is in addition to can be used as albumen
Matter drug, enzyme, vaccine and other biological product excellent activity protective agent.
Another kind of important freeze drying protectant is antifreeze protein.The most commonly used is that antifreeze glycoprotein (AFGP), I type are anti-
Freeze albumen (AFP I) and II type antifreeze protein (AFP II).Previous experiments of the present invention find three by constantly groping and practicing
Reasonable combination dosage can improve the redissolution effect of frozen-dried protective and freeze-dried powder significantly, and can be used in substitution has cell toxicant
DMSO.
5. freeze-dried powder of the invention and other beauty class product mixes use, the metabolism of Skin Cell can be accelerated, it is extensive
Complex radical floor cells are anti-oxidant and power of regeneration, prevent skin aging, increase skin elasticity and plumpness, reduce pore, promote light
Pool.Long-time service can get high skin improvement effects.
Specific embodiment
The present invention is further elaborated with below by embodiment.
The preparation of 1 human umbilical cord mesenchymal stem cells excretion body freeze-dried powder of embodiment
1.1 collect human umbilical cord's mescenchymal stem cell
A. collection healthy human body umbilical cord makees further cleaning to umbilical cord, then is cut with aseptic operation and be divided into umbilical cord cutting carefully
Cell；B. the fine tissue after shredding is digested umbilical cord tissue block 3 hours by pancreatin, the clostridiopetidase A etc. that debita spissitudo is added,
To differentiate monocyte, collects the monocyte differentiated and carry out adhere-wall culture with serum free medium, filled between acquisition umbilical cord
Matter stem cell.
1.2 carry out cell culture
Obtained mescenchymal stem cell single cell suspension will be collected and be placed in 37 DEG C, volume fraction 6.5%CO2In incubator
Culture, cell fusion collect the 4th generation cell as culture cell up to passing on after 90%；B. it chooses in culture cell and is in logarithm
Serum free medium DMEM/F12 is added in the cell in growth period, cultivates 72h, collects supernatant daily, and replace fresh culture
The supernatant for collecting culture is used for the extraction of excretion body by base.
The extraction of 1.3 mescenchymal stem cell excretion bodies
A. by the supernatant of collection at 4~6 DEG C, 20min is centrifuged with 12000r/min, supernatant is stayed, discards precipitating；b.
By supernatant obtained by a step at 4~6 DEG C, 60min is centrifuged with 28000r/min, supernatant is stayed, discards precipitating；C. b is walked
Rapid gained supernatant is centrifuged 70min with 12000r/min, discards supernatant liquid, it is dry to get mesenchyma to retain precipitating at 4~6 DEG C
Cell excretion body extracting solution.
1.4 extracting solution is lyophilized
A. freeze drying protectant is first added, and adjusting pH value is 6.5；Containing such as the following group in the every 100ml of freeze drying protectant
Point: 0.3~0.5g of ascorbic acid, 2.5~3.0g of dextran, trehalose 2g, 1~20g of antifreeze protein mixture, the tert-butyl alcohol 10
~20ml, wherein the antifreeze protein mixture is antifreeze glycoprotein (AFGP), I type antifreeze protein (AFP I) and II type are anti-
Freeze albumen (AFP II) mixture, three's mass ratio is 2:1:1；B. then be lyophilized: freeze-drying pressure be 20pa, temperature for-
25 DEG C, time 10h.
Embodiment 2
A kind of toner containing mescenchymal stem cell excretion body freeze-dried powder, which is characterized in that include following weight percentage
The component of ratio: mescenchymal stem cell excretion body freeze-dried powder (embodiment 1 is made) 5.0%, glycerol 1%, polyalcohols A 3%, sweet tea
Dish alkali 0.5%, panthenol 0.8%, dipotassium glycyrrhizinate 0.1%, other active components A 1.0%, polyethylene glycol -3 1.5%, surface
Activating agent A 0.3%, preservative A 0.2%, xanthan gum 0.15%, fragrance 0.03%, surplus are purified water；Wherein, described
Polyalcohols A is butanediol；The other active components A is the mixture of allantoin, niacinamide and Sodium Hyaluronate, San Zhezhi
Amount is 1:1:1；The surfactant A is the mixture of hydroxyethyl cellulose and Cremophor RH40, the two mass ratio
For 1:1；The preservative A is methyl hydroxybenzoate.Toner is prepared into according to conventional method in that art.
Embodiment 3
A kind of facial mask essence containing mescenchymal stem cell excretion body freeze-dried powder, which is characterized in that include following weight
The component of percentage: mescenchymal stem cell excretion body freeze-dried powder (embodiment 1 is made) 6.0%, glycerol 1%, polyalcohols B
3%, panthenol 0.8%, dipotassium glycyrrhizinate 0.05%, allantoin 0.3%, other active components B 1.0%, surfactant B
3.0%, preservative B 0.3%, xanthan gum 0.2%, fragrance 0.05%, and aminomethyl propanol is added, the aminomethyl propanol
Content, which is subject to, adjusts the facial mask essence pH value to 5.5-7, and surplus is purified water；The polyalcohols B is 1,3- third
Glycol；The other active components B is the mixture of niacinamide and Sodium Hyaluronate, and the two mass ratio is 1:1；The surface is living
Property agent B be -2 oleate of hydroxyethyl cellulose, isopropyl isostearate and polyglycereol mixture, three's mass ratio be 1:1:1；
The preservative B is Nipasol.Facial mask essence is prepared into according to conventional technical means in the art.
Embodiment 4
One kind containing mescenchymal stem cell freeze-dried powder excretion body moisturizing lotion, which is characterized in that mescenchymal stem cell freeze-drying
Powder (embodiment 1 is made) 8.0%, glycerol 1%, 1,3-PD 3%, panthenol 1%, tocopherol acetate 0.5%, other work
Property ingredient C 1.0%, dimethyl silicone polymer 1.5%, emulsifier A165 2%, isopropyl isostearate 0.5%, oils C
1.5%, cetostearyl alcohol 0.8%, preservative C 0.1%, xanthan gum 0.5%, EDTA-2Na 0.1%, and add three ethyl alcohol
Amine, the content of the triethanolamine are subject to the pH value for adjusting the lotion to 5.5-7, and surplus is purified water；Wherein, it is described its
His active constituent C is the mixture in dipotassium glycyrrhizinate, niacinamide and Parleam, and three's mass ratio is 1:1:1；It is described
Oils C is isohexadecane；The preservative C is methyl hydroxybenzoate.Lotion is prepared into according to conventional technical means in the art.
The comparison of 5 freeze drying protectant of embodiment
Experiment one: the comparison of freeze-drying time
1) it is formulated one (using the frozen-dried protective agent prescription of embodiment 1): containing as follows in the every 100ml of freeze drying protectant
Component: 0.3~0.5g of ascorbic acid, 2.5~3.0g of dextran, trehalose 2g, 1~20g of antifreeze protein mixture, the tert-butyl alcohol
10~20ml, wherein the antifreeze protein mixture is antifreeze glycoprotein (AFGP), I type antifreeze protein (AFP I) and II type
Antifreeze protein (AFP II) mixture, three's mass ratio are 2:1:1；
2) two are formulated: containing following component: 0.3~0.5g of ascorbic acid, the white egg of people in the every 100ml of freeze drying protectant
White 0.2~0.5ml, 2.5~3.0g of dextran, trehalose 2g, 0.02~0.04g of glycine, 0.05~0.15g of arginine,
10~20ml of the tert-butyl alcohol；
3) three are formulated: containing following component: DMSO10ml and fetal calf serum 10ml in the every 100ml of freeze drying protectant.
Formula one is respectively adopted and formula two is lyophilized, remaining step is identical, prepares freeze-dried powder respectively, carries out to the two
Compare, both discoveries performance difference is as follows:
As seen from the above table, the freeze-dried powder prepared using novel freeze drying protectant, in depositing for no fetal calf serum and DMSO
Under, freeze-drying time can be shortened, and reach compared with low moisture content, it is more lasting to illustrate to save, and after redissolution
It can obtain the stem cell excretion body as conventional liquid.
Embodiment 6- is directly smeared
1.1 pairs of patient's groupings
Volunteer 120 are recruited, is women, the age 25-50 years old, is divided into 2 groups.Wherein experimental group 60, wherein there is scar
Trace person 25, there are facial lines person 35；Control group 60, wherein having scar person 25, there are facial lines person 35.
1.2 experimental method
Experimental group: facial treatment milk prepared by embodiment 4 is smeared in face after face cleaning daily morning and evening, dosage is had one's face covered with uniform painting
Portion and slightly moist feeling are advisable and (about use 0.5~1), and 2 times a day, service life is 20 days；
Positive controls: using commercially available anti-wrinkle emulsion, smears after face cleaning in face daily morning and evening, and dosage is had one's face covered with uniform painting
Portion and slightly moist feeling are advisable 0.3~1.0ml, and 2 times a day, service life is 20 days.
1.3 experimental result
Observation experiment group volunteer's skin, scar improvement person reach 22, and the facial lines person of smoothing reaches 33, total effective
Rate reaches 92%.It can be seen that the present invention comfort trace, repair damage skin effect it is preferable.
Embodiment 7- is used in combination with water laser accunputure
1.1 pairs of patient's groupings
Volunteer 60 are recruited, is women, the age 25-50 years old, is divided into 2 groups.Wherein experimental group 30 have facial thin
Line；Control group 30, there are facial lines.
1.2 experimental method
Experimental group: facial mask essence and water laser accunputure prepared by the embodiment of the present invention 3 is mixed according to the ratio of 1:1, such as
The Essence of 2ml is mixed with the water laser accunputure of 2ml, after being prepared into mixed liquor, smears skin, and 2 times a day, service life is 60 days；
Positive controls: smearing skin using commercially available water laser accunputure, and 2 times a day, service life 2 times a day, used for 25 days
Period is 60 days.
1.3 experimental result
Observation experiment group volunteer's skin, the facial lines person of smoothing reach 25, and what skin became moistens glossy total effective
Rate reaches 83.3%.It can be seen that the present invention certain useful promotion skin metabolism, anti-aging, crease-resistant effect.
Claims (13)
1. a kind of composition for external application, which is characterized in that include mescenchymal stem cell excretion body freeze-dried powder, wherein between described
At least contain 25.0~35.0ng/g of fibronectin, vascular endothelial growth factor in mesenchymal stem cells excretion body freeze-dried powder
5.0~14.5ng/g, 5.5~15.5ng/g of fibrous bud Porcine HGF, 5.0~10.0ng/g of epithelial cell growth factor,
25.0~30.0ng/g of hepatocyte growth factor, 7.5~17.5ng/g of transforming growth factor, 0.5~5ng/g of sphingosine 1-phosphate
With 0.5~5ng/g of ceramide -1- phosphoric acid；Preferably, at least connect containing fine in the mescenchymal stem cell excretion body freeze-dried powder
Albumen 30.0ng/g, vascular endothelial growth factor 12.0ng/g, fibrous bud growth factor-21 2.5ng/g, epithelial cell
Growth factor 8.0ng/g, hepatocyte growth factor 26.0ng/g transforming growth factor 1 2.5ng/g, sphingosine 1-phosphate 4ng/g
With ceramide -1- phosphoric acid 3ng/g.
2. composition for external application according to claim 1, which is characterized in that the mescenchymal stem cell excretion body freeze-drying
The preparation method of powder is as follows, comprising the following steps:
S1 is collected: being collected mescenchymal stem cell, is obtained mescenchymal stem cell single cell suspension；Preferably, the mesenchyma is dry
Cell include mesenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, fat mesenchymal stem cell or
Muscle mesenchymal stem.
S2 culture: above-mentioned mescenchymal stem cell single cell suspension is inoculated into culture medium and is cultivated；
S3 is extracted: the above-mentioned culture medium containing mescenchymal stem cell being extracted and obtains mescenchymal stem cell excretion body extracting solution；
S4 freeze-drying: by above-mentioned excretion body extracting solution be lyophilized to get.
3. composition for external application according to claim 2, which is characterized in that cell culture processes described in step S2
The following steps are included:
A. will collect obtained mescenchymal stem cell single cell suspension be placed in 37 DEG C, volume fraction be 4%~8%CO2In incubator
Culture, cell fusion pass on after reaching 80%-95%, collect 3-5 for cell as culture cell；Preferably, the CO2Body
Fraction is 5~6.5%；Cell fusion is 90%；Cell is the 4th generation cell；
B. the cell in logarithmic growth phase in selection culture cell, addition serum free medium DMEM/F12, culture 48~
96h collects supernatant daily, and replaces fresh culture medium, and the supernatant for collecting culture is used for the extraction of excretion body；It is preferred that
, the incubation time is 72h.
4. composition for external application according to claim 2, which is characterized in that extraction step described in step S3 is specific
Include:
A. by the supernatant collected in step S2 at 4~6 DEG C, 20~30min is centrifuged with 10000~15000r/min, is stayed
Clear liquid discards precipitating；
B. by supernatant obtained by a step at 4~6 DEG C, 60~80min is centrifuged with 15000~30000r/min, stays supernatant,
Discard precipitating；
C. by supernatant obtained by b step at 4~6 DEG C, 60~80min is centrifuged with 10000~15000r/min, is discarded supernatant
Liquid retains precipitating to get mescenchymal stem cell excretion body extracting solution.
5. composition for external application according to claim 2, which is characterized in that freeze drying process described in step S4 includes
Following steps:
A. freeze drying protectant is added: freeze drying protectant is added in Xiang Shangshu mescenchymal stem cell excretion body extracting solution, and adjusts pH value
It is 6.5；In the every 100ml of freeze drying protectant contain following component: 0.3~0.5g of ascorbic acid, dextran 2.5~
3.0g, trehalose 2g, 1~20g of antifreeze protein mixture, 10~20ml of the tert-butyl alcohol；Preferably, the antifreeze protein mixture
For antifreeze glycoprotein, I type antifreeze protein and II type antifreeze protein mixture, three's mass ratio is 2:1:1；
B. be lyophilized: freeze-drying pressure is 10~30pa, and temperature is -25~-30 DEG C, and the time is 10~12h.
6. described in any item composition for external application according to claim 1~5, which is characterized in that also containing can in cosmetics
The carrier of receiving, including at least two or more in antioxidant, stabilizer, solubilizer, pigment, fragrance.
7. composition for external application according to claim 6, which is characterized in that the dosage form of the composition for external application is molten
Liquid, lotion, gel, creams, lotion, pulvis, soap, the detergent containing surfactant, any one in finish.
8. composition for external application according to claim 7, which is characterized in that the composition for external application is makeup
Water, facial mask essence, lotion, gel, creams, essential oil, cleanser, cleaning foam, clean water or spray.
9. composition for external application according to claim 7, which is characterized in that the composition for external application is makeup
Water, the component comprising following weight percentage: mescenchymal stem cell excretion body freeze-dried powder 5.0~8.0%, glycerol 1%, polyalcohol
Class A 3~5%, glycine betaine 0.5%, panthenol 0.8%, dipotassium glycyrrhizinate 0.05~0.1%, other active components A 0.11~
1.0%, polyethylene glycol -3 1.0~1.5%, surfactant A 0.25~0.3%, preservative A 0.01~0.25%, xanthan
Glue 0-0.15%, fragrance 0.03%, surplus are purified water；Wherein, the polyalcohols A be butanediol, 1,2- pentanediol and
One of dipropylene glycol is a variety of；The other active components A be one of allantoin, niacinamide and Sodium Hyaluronate or
It is a variety of；The surfactant A be one of hydroxyethyl cellulose, polysorbate -20 and Cremophor RH40 or
It is a variety of；The preservative A is methyl hydroxybenzoate and/or Phenoxyethanol.
10. composition for external application according to claim 7, which is characterized in that the composition for external application is facial mask
Essence, the component comprising following weight percentage: mescenchymal stem cell excretion body freeze-dried powder 5.0~8.0%, glycerol 1% is more
First alcohols B 3~5%, panthenol 0.8%, dipotassium glycyrrhizinate 0.05~0.1%, allantoin 0.2~0.3%, other active components B
0.5-2.0%, surfactant B 0.05-3.6%, preservative B 0.25-0.45%, xanthan gum 0.2-0.4%, fragrance
0.05%, and add aminomethyl propanol, the content of the aminomethyl propanol is to adjust the facial mask essence pH value to 5.5-7
Standard, surplus are purified water；The polyalcohols B is butanediol and/or 1,3- propylene glycol；The other active components B is cigarette
One of amide, Sodium Ascorbyl Phosphate, tocopherol acetate and Sodium Hyaluronate are a variety of；The surfactant B
For one of hydroxyethyl cellulose, isopropyl isostearate, -2 oleate of polyglycereol and Natrulon H-10 oleate or a variety of；
The preservative B is one of Nipasol, methyl hydroxybenzoate or Phenoxyethanol or a variety of.
11. composition for external application according to claim 10, which is characterized in that the composition for external application is cream
Liquid, the component comprising following weight percentage: mescenchymal stem cell excretion body freeze-dried powder 5.0~8.0%, glycerol 1%, 1,3- third
Glycol 3-5%, panthenol 1%, tocopherol acetate 0.5%, other active components C 0.1-2.0%, dimethyl silicone polymer
0.5-2%, emulsifier A165 0.5-2%, isopropyl isostearate 0.5%, oils C 0.2-3%, cetostearyl alcohol 0.8%,
Preservative C 0.1%, xanthan gum 0.5%, EDTA-2Na 0.1%, and add triethanolamine, the content of the triethanolamine with
Subject to the pH value to 5.5-7 for adjusting the lotion, surplus is purified water；Wherein, the other active components C is glycyrrhizic acid two
One of potassium, niacinamide and Parleam are a variety of；The oils C is dormant oil and/or isohexadecane；The preservative
C is methyl hydroxybenzoate and/or Nipasol.
12. the composition for external application that a kind of any one of claim 1-11 composition for external application repairs damage skin in preparation
In purposes.
13. the skin that a kind of any one of claim 1-11 composition for external application promotes skin metabolism or anti-aging in preparation
Application in skin topical composition.
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| CN111281887A (en) * | 2020-03-03 | 2020-06-16 | 中山大学 | Thermo-sensitive hydrogel compound suitable for carrying human umbilical cord Wharton's jelly mesenchymal stem cells and application thereof |
| CN111407718A (en) * | 2020-03-30 | 2020-07-14 | 北京神秘美医疗美容诊所有限责任公司 | Cell regeneration and repair essence for medical cosmetology and preparation method thereof |
| CN113209135A (en) * | 2021-04-23 | 2021-08-06 | 奥启(深圳)创投科技有限公司 | Mesenchymal stem cell exosome preparation for delaying skin aging |
| CN113797151A (en) * | 2021-10-09 | 2021-12-17 | 四川伍衍生物科技有限公司 | Repairing gel containing mesenchymal stem cell exosomes and preparation method thereof |
| CN113925825A (en) * | 2021-11-18 | 2022-01-14 | 张�杰 | Facial essence and preparation method thereof |
| CN114681386A (en) * | 2022-03-16 | 2022-07-01 | 和携科技有限公司 | Animal-derived exosome composition and application thereof in preparation of anti-aging and anti-allergic skin preparation |
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| CN115369073A (en) * | 2022-08-23 | 2022-11-22 | 北京翌宁细胞生物技术有限公司 | Preparation method of epidermal stem cell exosome |
| CN116531323A (en) * | 2023-07-06 | 2023-08-04 | 北京恩康医药有限公司 | Stabilizer composition for exosome products and application thereof |
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| CN113209135A (en) * | 2021-04-23 | 2021-08-06 | 奥启(深圳)创投科技有限公司 | Mesenchymal stem cell exosome preparation for delaying skin aging |
| CN113797151A (en) * | 2021-10-09 | 2021-12-17 | 四川伍衍生物科技有限公司 | Repairing gel containing mesenchymal stem cell exosomes and preparation method thereof |
| CN113797151B (en) * | 2021-10-09 | 2023-10-31 | 四川伍衍生物科技有限公司 | Repairing gel containing mesenchymal stem cell exosomes and preparation method thereof |
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| CN114681386A (en) * | 2022-03-16 | 2022-07-01 | 和携科技有限公司 | Animal-derived exosome composition and application thereof in preparation of anti-aging and anti-allergic skin preparation |
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