CN108578264A - Placental hormone and its preparation, application - Google Patents
Placental hormone and its preparation, application Download PDFInfo
- Publication number
- CN108578264A CN108578264A CN201810508526.9A CN201810508526A CN108578264A CN 108578264 A CN108578264 A CN 108578264A CN 201810508526 A CN201810508526 A CN 201810508526A CN 108578264 A CN108578264 A CN 108578264A
- Authority
- CN
- China
- Prior art keywords
- supernatant
- placental hormone
- preparation
- obtains
- placental
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
-
- 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
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
-
- 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
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
- A61K8/982—Reproductive organs; Embryos, Eggs
-
- 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
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Reproductive Health (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
A kind of placental hormone of present invention offer and its preparation, application, the preparation method of the placental hormone include：Fresh animal placenta tissue and/or umbilical cord tissue are selected, is cleaned, chopping is placed in ethyl sodium buffer solution and is homogenized, and centrifuges, and obtains supernatant A and precipitation；Supernatant A is taken, ethyl alcohol, mixing is added, centrifugation obtains supernatant B；Rotary evaporation is carried out to supernatant B, obtains yolk yellow dope；The yolk yellow dope is taken, solution is prepared into, is centrifuged, supernatant C is obtained；Precipitation is taken, is dissolved in water, is digested, centrifugation obtains supernatant D；Supernatant C, supernatant D are mixed, placental hormone semifinished product is obtained；To the filtering of placental hormone semifinished product, anion-exchange chromatography, Peak Activity is collected, obtains placental hormone.The present invention can effectively avoid the change of active ingredient native conformation, additionally it is possible to ensure that active ingredients skin makeup obvious effect, the effects such as collagen, polypeptide, a variety of growth factors, the stem cell biological activated protein in retaining in a organized way in the placental hormone that extraction obtains are lasting.
Description
Technical field
The present invention relates to biotechnologies, more particularly to placental hormone and its preparation, application.
Background technology
Skin aging is by external factor and internal factor process extremely complex caused by common.Cause skin aging
External factor includes life style, nutrition condition, environmental factor etc..Wherein, intensive ultraviolet, dust, haze, drying are in daylight
Lead to the essential environmental factors of skin aging.Internal factor includes mainly：Cell telomere is gradually shortened with age, and thin
Born of the same parents' oxidative damage but gradually accumulates；The expression of intracellular various kinds of cell growth factor declines；Collagenous fibres aggregate velocity by
Decrescence slow, skin elasticity is deteriorated；Veins beneath the skin atrophy, so as to cause skin blood supply insufficiency；Skin Cell lacks nutrition, and update subtracts
There are the senile symptoms such as wrinkle, color spot in weak, skin.
With the improvement of living standards, more and more people start to focus on the maintaining skin such as He Jiankang, it is desirable that also more next
It is higher.In current cosmetology industry, traditional beauty has been unable to meet demand, a variety of natural extract component beauty
Cosmetic is on the occasion of prosperous.Although current cosmetics are dazzling, existing cosmetics and skincare product, nutritional ingredient often only stops
In skin surface, deep skin reparation can not be penetrated into, effect is difficult to persistently.And due to wherein chemical addition agent, preservative etc.
The irritation of ingredient leads to the allergic phenomenas such as red swelling of the skin, fash often occur.In addition, the beauty skin care product of listing, effect
It is single, more money products are often required to use, it is cumbersome, it is costly.
Placental hormone is to be also known as " placenta peptide " by the general name with immunocompetent micromolecular compound that placenta extracts.In
Medical knowledge opinion thinks, placenta is dry, salty, return lung, kidney channel.Function be tonifying Qi, blood-nourishing, strengthening the essence, can be used as nourishing, beauty treatment, it is strong it
Function.Its effect mainly has the following：There is health-care effect to skin, have a significant impact to immunocompetence and locomitivity, resist
Fatigue effect, anti-aging effects, immunization of cell.The prior art indicate that although placental hormone can not can prolong to anti-aging
The generation of slow aging, is increasingly paid attention to by scientist.
Prior art discloses the methods and purposes of extraction of activated micromolecular sheep placenta in a kind of embryo from sheep.This method will
After fresh sheep embryo chopping plus homogenate is made in distilled water, and in low temperature multigelation, filtering, filtered solution is made after filtrate is boiled
And sediment, then respectively filtered fluid, sediment handle again and liquid merges by treated, finally obtains molecular weight
The sheep placenta between 3000~6000 is controlled, can be used for preparing external application wrinkle reduction, beautifying skin articles for use.But this is existing
During technology extracts sheep placenta from embryonic tissue, the change that high temperature would generally cause active protein peptide chain space structure makes
It loses natural structure, can also cause the loss of active ingredient, to seriously affect final products activity, Simultaneous purification step mistake
In simple, finished product purity is not high.
It would therefore be highly desirable to provide a kind of change that can effectively avoid active ingredient native conformation, the tire that active ingredient is lost in
Disk element preparation method.
Invention content
Based on this, an object of the present invention is held for active ingredient loss, natural structure in placental hormone preparation process
The problem of malleable, provides a kind of preparation method of placental hormone.
The purpose of the present invention is what is be achieved through the following technical solutions：
A kind of preparation method of placental hormone, the preparation method include：
In vitro fresh animal placenta tissue and/or umbilical cord tissue are selected, is cleaned, chopping is placed in acetic acid-sodium acetate buffer solution
Middle homogenate, centrifugation obtain supernatant A and precipitation；
The supernatant A is taken, ethyl alcohol, mixing is added, centrifugation obtains supernatant B；Rotary evaporation is carried out to supernatant B, obtains yolk
Color dope；The yolk yellow dope is taken, solution is prepared into, is centrifuged, supernatant C is obtained；
The precipitation is taken, is dissolved in water, is digested, centrifugation obtains supernatant D；
The supernatant C, supernatant D are mixed, placental hormone semifinished product is obtained；
To placental hormone semifinished product filtering, anion-exchange chromatography, Peak Activity is collected, obtains placental hormone.
In wherein some embodiments, obtain supernatant A the step of in, a concentration of the 45 of the acetic acid-sodium acetate buffer solution
~60mmoL/L, pH is 3.5~4.5；In the step of obtaining supernatant B, the temperature of ethyl alcohol is -25~-15 DEG C, when being added ethyl alcohol
Environment temperature is 1~5 DEG C.
In wherein some embodiments, obtain supernatant D the step of in, it is described enzymolysis using trypsase, the pancreas
The working concentration of protease is 0.05~0.15%, and the time of enzymolysis is 6~8h.
In wherein some embodiments, in the step of obtaining supernatant A, the condition of centrifugation is 1~5 DEG C, 6500~7000r/
Min, 25~35min；In the step of obtaining supernatant B, the condition of centrifugation is 1~5 DEG C, 6500~7000r/min, 25~35min；
In the step of obtaining supernatant C, the condition of centrifugation is 15000~18000r/min, 15~25min；In the step of obtaining supernatant D,
The condition of the centrifugation be 1~5 DEG C, 6500~7000r/min, 25~35min.
In wherein some embodiments, in the step of obtaining yolk yellow dope, bath temperature 35 that rotary evaporation uses
~45 DEG C；The filtering uses 0.2~0.45 μm of membrane filtration.
It is a further object of the present invention to provide a kind of placental hormones that above-mentioned preparation method obtains.
A kind of purposes it is a further object of the present invention to provide above-mentioned placental hormone as beauty product active ingredient.
Another object of the present invention is to provide a kind of composition with beauty functions, includes above-mentioned placental hormone.
In wherein some embodiments, the composition includes each raw material of following mass percentage：
Wherein, the placental hormone refined liquid is the normal saline solution for the placental hormone that mass concentration is 5~15%.
In wherein some embodiments, the preparation of the stem cell biological activated protein includes：
Fresh animal placenta tissue and/or umbilical cord tissue are selected, cleans, is cut into fragment of tissue；
Fragment of tissue is taken, DMF12 culture solution cultures are added, obtains mescenchymal stem cell；
Mescenchymal stem cell is taken, DMF12 culture solution cultures are added, culture is collected after culture；It removes in culture
Cell fragment, ultrafiltration, drying, i.e. stem cell biological activated protein.
The ultrafiltration includes：First using the PBS solution that mass concentration is 3~8% as purification solution 20~30h of ultrafiltration, purifying
It is 2~3 times that solution, which replaces number,；Again using the PEG6000 solution that mass concentration is 15~25% as 3~5h of purification solution ultrafiltration.
In wherein some embodiments, the ultrafiltration includes：The PBS solution for being first 5% using mass concentration is purification solution
For 24 hours, it is 2~3 times that purification solution, which replaces number, for ultrafiltration；Surpass again by purification solution of the PEG6000 solution that mass concentration is 20%
Filter 3~5h.
In wherein some embodiments, the pH adjusting agent is triethanolamine.
Compared with prior art, the present invention has following advantageous effect：
Inventor is molten using acetic acid-sodium acetate buffer solution after chopping fresh animal placenta tissue and/or umbilical cord tissue
Liquid is homogenized, is centrifuged, and supernatant precipitation is obtained；For supernatant, cooperation using ethyl alcohol reprocess and to obtained by the processing
The solution rotating of dope is evaporated, and corresponding evaporation supernatant is obtained；The evaporation supernatant with precipitation digest gained enzymolysis supernatant mix,
Purifying, that is, obtain placental hormone.In the preparation process of the placental hormone, inventor is homogenized using acetic acid-sodium acetate buffer solution solution
And coordinate and follow-up particular procedure is carried out to supernatant using ethyl alcohol, it is homogenized and relative to conventional prepared using distilled water by freezing repeatedly
Melt, heat for the supernatant that centrifuges again precipitates to prepare placental hormone, animal placenta tissue and/or umbilical cord group can be made
It knits and is fully hydrolyzed, maximum discharges active ingredient；The change of active ingredient native conformation is effectively avoided simultaneously, additionally it is possible to ensure to carry
Collagen, polypeptide, a variety of growth factors, stem cell biological activated protein in retaining in a organized way in the placental hormone obtained etc. are effective
Ingredient can be achieved at the same time skin-whitening, go wrinkle, tender skin, nti-freckle etc. when the placental hormone is used as the active ingredient of beauty product
Multiple functions, infiltration deep skin reparation, obvious effect, effect are lasting；And it is simple for process.
Figure of description
Fig. 1 is the placental hormone preparation flow figure of the embodiment of the present invention；
Fig. 2 is the chromatography peak figure for the placental hormone that the embodiment of the present invention is prepared.
Specific implementation mode
Below in conjunction with specific embodiment, the present invention is described in further detail.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
People, molecular cloning：Laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) institute in
The condition stated, or according to the normal condition proposed by manufacturer.Used various common chemical reagent, are commercially available in embodiment
Product.
Unless otherwise defined, all technical and scientific terms used in the present invention and the technical field for belonging to the present invention
The normally understood meaning of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term used in the present invention " and/5 or " includes one or more relevant institutes
Any and all combinations of list of items.
Animal placenta of the present invention does not include Human embryo, specifically such as in vitro sheep embryo.
The stem cell biological activated protein that following embodiment is related to, obtains as follows：
In vitro animal placenta tissue is selected, is cleaned, blood vessel and mucosal tissue is removed, is cut into fragment of tissue；
Fragment of tissue is taken, DMF12 culture solution cultures are added, obtains mescenchymal stem cell；
Mescenchymal stem cell is taken, DMF12 culture solution cultures are added, culture is collected after culture；It removes in culture
Cell fragment, ultrafiltration, drying, i.e. stem cell biological activated protein；
The ultrafiltration includes：First using the PBS solution that mass concentration is 3~8% as purification solution 20~30h of ultrafiltration, purifying
It is 2~3 times that solution, which replaces number,；Again using the PEG6000 solution that mass concentration is 15~25% as 3~5h of purification solution ultrafiltration.
Preferably, the ultrafiltration includes：First using the PBS solution that mass concentration is 5% as purification solution ultrafiltration for 24 hours, purification solution replace
Number is 2~3 times；Again using the PEG6000 solution that mass concentration is 20% as 3~5h of purification solution ultrafiltration.
Concrete operations are as follows：
Step (1) selects fresh and healthy Goat Placenta tissue 100mL or umbilical cord tissue 10cm long by screening；
Placenta tissue is fully cleaned and is cut into small pieces by step (2), is removed blood vessel and mucosal tissue, is shredded into 5mm3；
Step (3), fragment of tissue is attached in 10cm sterile petri dish, and DMF12 culture solutions are added, are placed in the two of 5%
Culture can be passed on to cell in carbonoxide incubator, routinely reach the 3rd generation, as mescenchymal stem cell；
Step (4) chooses the 3rd generation active mescenchymal stem cell, and routine culture to growth density is 60%, then
It carries out changing liquid processing after being cleaned with PBS solution；The conventional culture conditions of the present invention refer to that the culture medium used is cultivated for DMF12
Base, cultivation temperature are 37 DEG C, and cultivating system contains 5% carbon dioxide；
Step (5) collects the culture medium of mescenchymal stem cell, by culture medium supernatant efficient filter mistake after culture
Filter removes cell fragment, is subsequently placed in high-performance ultrafiltration bag, and purification solution selects 5%PBS, low-temperature treatment for 24 hours, during which to replace
Purification solution 2~3 times；
Step (6), high-performance ultrafiltration bag is placed in 20%PEG6000 and continues with 3~5h, stem cell as after purification
Biological activity protein concentrate freeze-dried can preserve.
Embodiment 1
The present embodiment provides a kind of placental hormone and preparation method thereof, which includes the following steps (referring to Fig. 1)：
Step (1) selects the fresh and healthy Goat Placenta tissue 100mL by screening；
Placenta tissue is fully cleaned and is cut into small pieces by step (2), removes blood vessel and mucosal tissue, chopping are placed on
Homogenate in 100mL 50mmoL/L acetic acid-sodium acetate buffer solutions (PH4.0), then refrigerated centrifuge takes supernatant to precipitate, upper honest and upright and thrifty
150mL；In the step, the condition of centrifugation is 4 DEG C, 6600r/min, 30min；
The ethyl alcohol (- 20 DEG C of precoolings) of 6 times of supernatant volumes is slowly added dropwise into supernatant, process is added dropwise at 4 DEG C for step (3)
In be stirred continuously, supernatant is taken after centrifugation；In the step, the condition of centrifugation is 4 DEG C, 6600r/min, 30min；
Step (4) is evaporated supernatant with rotary evaporator, and 40 DEG C of bath temperature obtains yolk yellow dope.It is given birth to 12.0mL
Salt water dissolution is managed, centrifugation takes supernatant；In the step, the condition of centrifugation is 16000r/min, 20min；
4 times of weight deionized waters are added in step (5), the precipitation in step (2), and pH8.0 is adjusted in stirring, and 0.1% pancreas egg is added
White enzyme hydrolyzes 7 hours, refrigerated centrifuge, after take supernatant to be mixed with the supernatant in step (4), as placental hormone semifinished product；The step
In, the condition of centrifugation is 4 DEG C, 6600r/min, 30min；
Step (6), 0.2 μm of membrane filtration of placental hormone semifinished product in step (5) further use DE-52 anion exchanges
Chromatography collects all Peak Activities, obtains placental hormone.The chromatography peak figure of placental hormone obtained by the present embodiment is shown in Fig. 2, according to fig. 2, peak face
Product represents greatly Tot Prot height, and it is more that peak number amount represents active peptide type more.
The obtained gained placental hormone of the present embodiment is configured to the placental hormone that mass concentration is 10% with physiological saline and refines
Liquid freeze-dried can also preserve.
Embodiment 2
The present embodiment provides a kind of placental hormone and preparation method thereof, which includes the following steps (referring to Fig. 1)：
Step (1) selects the fresh and healthy Goat Placenta tissue 100mL by screening；
Placenta tissue is fully cleaned and is cut into small pieces by step (2), removes blood vessel and mucosal tissue, chopping are placed on
Homogenate in 100mL 45mmoL/L acetic acid-sodium acetate buffer solutions (pH3.5), then refrigerated centrifuge takes supernatant to precipitate, upper honest and upright and thrifty
150mL；In the step, the condition of centrifugation is 1 DEG C, 6500r/min, 25min；
The ethyl alcohol (- 25 DEG C of precoolings) of 6 times of supernatant volumes is slowly added dropwise into supernatant, process is added dropwise at 1 DEG C for step (3)
In be stirred continuously, supernatant is taken after centrifugation；In the step, the condition of centrifugation is 1 DEG C, 6500r/min, 25min；
Step (4) is evaporated supernatant with rotary evaporator, and 35 DEG C of bath temperature obtains yolk yellow dope.It is given birth to 12.0mL
Salt water dissolution is managed, centrifugation takes supernatant；In the step, the condition of centrifugation is 15000r/min, 15min；
3 times of weight deionized waters are added in step (5), the precipitation in step (2), and pH7.0 is adjusted in stirring, and 0.05% pancreas is added
Protease hydrolyzes 6 hours, refrigerated centrifuge, after take supernatant to be mixed with the supernatant in step (4), as placental hormone semifinished product；The step
In rapid, the condition of centrifugation is 1 DEG C, 6500r/min, 25min；
Step (6), 0.2 μm of membrane filtration of placental hormone semifinished product in step (5) further use DE-52 anion exchanges
Chromatography collects all Peak Activities, obtains placental hormone.
The obtained gained placental hormone of the present embodiment is configured to the placental hormone that mass concentration is 5% with physiological saline and refines
Liquid freeze-dried can preserve.
Embodiment 3
The present embodiment provides a kind of placental hormone and preparation method thereof, which includes the following steps (referring to Fig. 1)：
Step (1) selects the fresh and healthy Goat Placenta tissue 100mL by screening；
Placenta tissue is fully cleaned and is cut into small pieces by step (2), removes blood vessel and mucosal tissue, chopping are placed on
Homogenate in 100mL 60mmoL/L acetic acid-sodium acetate buffer solutions (pH4.5), then refrigerated centrifuge takes supernatant to precipitate, upper honest and upright and thrifty
150mL；In the step, the condition of centrifugation is 5 DEG C, 7000r/min, 35min；
The ethyl alcohol (- 15 DEG C of precoolings) of 6 times of supernatant volumes is slowly added dropwise into supernatant, process is added dropwise at 5 DEG C for step (3)
In be stirred continuously, supernatant is taken after centrifugation；In the step, the condition of centrifugation is 5 DEG C, 7000r/min, 35min；
Step (4) is evaporated supernatant with rotary evaporator, and 45 DEG C of bath temperature obtains yolk yellow dope.It is given birth to 12.0mL
Salt water dissolution is managed, centrifugation takes supernatant；In the step, the condition of centrifugation is 18000r/min, 25min；
5 times of weight deionized waters are added in step (5), the precipitation in step (2), and pH9.0 is adjusted in stirring, and 0.15% pancreas is added
Protease hydrolyzes 8 hours, refrigerated centrifuge, after take supernatant to be mixed with the supernatant in step (4), as placental hormone semifinished product；The step
In rapid, the condition of centrifugation is 5 DEG C, 7000r/min, 35min；
Step (6), 0.2 μm of membrane filtration of placental hormone semifinished product in step (5) further use DE-52 anion exchanges
Chromatography collects all Peak Activities, obtains placental hormone.
The obtained gained placental hormone of the present embodiment is configured to the placental hormone that mass concentration is 15% with physiological saline and refines
Liquid freeze-dried can preserve.
Comparative example 1
This example is the comparative example of embodiment 1, and difference is step (2) and step (3)：
Goat Placenta tissue is fully cleaned and is cut into small pieces by step (2), removes blood vessel and mucosal tissue, chopping are placed on
Homogenate in 100mL 50mmoL/L PBS buffer solution (PH7.0), then refrigerated centrifuge takes supernatant to precipitate, upper honest and upright and thrifty 150mL；It should
In step, the condition of centrifugation is 4 DEG C, 6600r/min, 30min；
The ultra-pure water (- 20 DEG C of precoolings) of 6 times of supernatant volumes is slowly added dropwise into supernatant, was added dropwise at 4 DEG C for step (3)
It is stirred continuously in journey, supernatant is taken after centrifugation；In the step, the condition of centrifugation is 4 DEG C, 6600r/min, 30min.
As a result, centrifuged deposit object is excessive (hydrolyzing insufficient), active ingredient does not precipitate, prepares placenta fibroin
Containing extremely low.
Comparative example 2
This example is the comparative example of embodiment 1, and difference is step (2) and step (3)：
Goat Placenta tissue is fully cleaned and is cut into small pieces by step (2), removes blood vessel and mucosal tissue, chopping are placed on
Homogenate in 100mL 40mmoL/L acetic acid-sodium acetate buffer solutions (PH5.0), then refrigerated centrifuge takes supernatant to precipitate, upper honest and upright and thrifty
150mL；In the step, the condition of centrifugation is 4 DEG C, 6600r/min, 30min；
The ethyl alcohol (- 5 DEG C of precoolings) of 6 times of supernatant volumes is slowly added dropwise into supernatant, process is added dropwise at 4 DEG C for step (3)
In be stirred continuously, supernatant is taken after centrifugation；In the step, the condition of centrifugation is 4 DEG C, 6600r/min, 30min.
As a result, centrifuged deposit object is relatively more (hydrolysis charge less point), active ingredient does not precipitate completely, prepares placenta
Fibroin content is relatively low.
The placental hormone refined liquid and Stem Cell Activity albumen being prepared with above each example prepare the group with skin makeup
Object is closed, composition of raw materials is shown in Table 1.
Performance test
The whitening of composition described in upper table 1, anti-wrinkle, freckle effect are tested, including handled as follows：
(1) experimental products：Composition 1 in table 1 is to composition 6；
(2) experimental method：40 25~45 years old women are randomly divided into 2 groups, every group of 20 people.Above-mentioned composition is used respectively
The back of the hand, the facial skin of experimental subjects are smeared, then massages masking liquid position 10 minutes.Once a day, it is used continuously 10 times.
Last evaluation experimental group and the whitening of control group, anti-wrinkle, freckle effect：Significant effect refers to whitening, removes wrinkle, timid spot
Effect reaches 50% or more；Effect generally refers to whitening, goes wrinkle, timid spot effect between 20~50%；Effect unobvious refer to
Whitening goes wrinkle, timid spot below 20%.
The results are shown in table below.
According to table 2, the placenta stem-cell biological agent containing placental hormone refined liquid, stem cell biological activated protein ingredient has
Significant whitening, anti-wrinkle, freckle effect.
Above example composition of the present invention is the placenta stem-cell biological agent for beauty and skin care.The stem cell of addition
Bioactive substance can promote the formation of veins beneath the skin, repair subcutaneous blood microcirculqtory system, improve the microenvironment of cell growth.
Stem cell effective active composition of the above example composition of the present invention to be extracted from placenta and/or placenta (umbilical cord)
For active ingredient, the beauty products such as facial mask, spraying, importing can be prepared into, multiple effect is easy to operate.
It is active that the present invention completely remains collagen, polypeptide, a variety of growth factors, stem cell biological in placenta and stem cell
The active ingredients such as albumen can be achieved at the same time skin-whitening, go the multiple functions such as wrinkle, tender skin, nti-freckle, penetrate into deep skin and repair
Again, obvious effect, effect are lasting.Also, the present invention is natural extract component, without containing any chemical cosmetic agents, no allergy,
High safety uses simplicity.
The present invention is purified through multistep techniques and is made, and operating method is simply controllable；Placental hormone stem cell biological in the present invention
Preparation use scope is extensive, and usability methods are various, easy to operate effective.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of preparation method of placental hormone, which is characterized in that the preparation method includes：
Fresh animal placenta tissue and/or umbilical cord tissue to be selected, is cleaned, chopping is placed in acetic acid-sodium acetate buffer solution and is homogenized,
Centrifugation obtains supernatant A and precipitation；
The supernatant A is taken, ethyl alcohol, mixing is added, centrifugation obtains supernatant B；Rotary evaporation is carried out to supernatant B, is obtained yolk yellow viscous
Thick object；The yolk yellow dope is taken, solution is prepared into, is centrifuged, supernatant C is obtained；
The precipitation is taken, is dissolved in water, is digested, centrifugation obtains supernatant D；
The supernatant C, supernatant D are mixed, placental hormone semifinished product is obtained；
To placental hormone semifinished product filtering, anion-exchange chromatography, Peak Activity is collected, obtains placental hormone.
2. the preparation method of placental hormone according to claim 1, which is characterized in that in the step of obtaining supernatant A, the second
A concentration of 45~60mmoL/L, the pH of acid-sodium acetate buffer are 3.5~4.5；In the step of obtaining supernatant B, the temperature of ethyl alcohol
It it is -25~-15 DEG C, environment temperature when ethyl alcohol is added is 1~5 DEG C.
3. the preparation method of placental hormone according to claim 1, which is characterized in that in the step of obtaining supernatant D, the enzyme
For solution using trypsase, the working concentration of the trypsase is 0.05~0.15%, and the time of enzymolysis is 6~8h.
4. the preparation method of the placental hormone according to claims 1 to 3, which is characterized in that in the step of obtaining supernatant A, from
The condition of the heart be 1~5 DEG C, 6500~7000r/min, 25~35min；In the step of obtaining supernatant B, the condition of centrifugation is 1~5
DEG C, 6500~7000r/min, 25~35min；In the step of obtaining supernatant C, the condition of centrifugation be 15000~18000r/min,
15~25min；In the step of obtaining supernatant D, the condition of the centrifugation is 1~5 DEG C, 6500~7000r/min, 25~35min.
5. the preparation method of placental hormone according to any one of claims 1 to 3, which is characterized in that obtain yolk yellow sticky
In the step of object, 35~45 DEG C of the bath temperature of rotary evaporation use；The filtering uses 0.2~0.45 μm of membrane filtration.
6. the placental hormone that any one of claim 1 to 5 preparation method obtains.
7. purposes of the placental hormone as beauty product active ingredient described in claim 6.
8. a kind of composition with beauty functions, which is characterized in that include the placental hormone described in claim 6.
9. composition according to claim 8, which is characterized in that the composition includes each original of following mass percentage
Material：
Wherein, the placental hormone refined liquid is the normal saline solution for the placental hormone that mass concentration is 5~15%.
10. composition according to claim 9, which is characterized in that the preparation of the stem cell biological activated protein includes：
Fresh animal placenta tissue and/or umbilical cord tissue are selected, cleans, is cut into fragment of tissue；
Fragment of tissue is taken, DMF12 culture solution cultures are added, obtains mescenchymal stem cell；
Mescenchymal stem cell is taken, DMF12 culture solution cultures are added, culture is collected after culture；It removes thin in culture
Born of the same parents' fragment, ultrafiltration, drying, i.e. stem cell biological activated protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810508526.9A CN108578264A (en) | 2018-05-24 | 2018-05-24 | Placental hormone and its preparation, application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810508526.9A CN108578264A (en) | 2018-05-24 | 2018-05-24 | Placental hormone and its preparation, application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108578264A true CN108578264A (en) | 2018-09-28 |
Family
ID=63629048
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810508526.9A Pending CN108578264A (en) | 2018-05-24 | 2018-05-24 | Placental hormone and its preparation, application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108578264A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111588737A (en) * | 2020-04-17 | 2020-08-28 | 焕生汇生物基因技术(北京)有限公司 | Efficient preparation method of placenta extract containing multiple growth factors |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896268A (en) * | 2006-06-27 | 2007-01-17 | 扬州大学 | Extraction of activated micromolecular sheept germine from sheep embryo and its use |
| CN102586182A (en) * | 2010-12-17 | 2012-07-18 | 张正前 | Preparation and application of human-stem-cell-secreted bioactive factor and lysis solution |
| CN107375334A (en) * | 2017-08-02 | 2017-11-24 | 广州准优生物科技有限公司 | Intacellin and its preparation method and application |
| CN107541486A (en) * | 2017-08-23 | 2018-01-05 | 奥尔文（深圳）生物科技有限公司 | A kind of placenta Subaerial blue green algae active factors of beauty and skin care and its preparation and application |
-
2018
- 2018-05-24 CN CN201810508526.9A patent/CN108578264A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896268A (en) * | 2006-06-27 | 2007-01-17 | 扬州大学 | Extraction of activated micromolecular sheept germine from sheep embryo and its use |
| CN102586182A (en) * | 2010-12-17 | 2012-07-18 | 张正前 | Preparation and application of human-stem-cell-secreted bioactive factor and lysis solution |
| CN107375334A (en) * | 2017-08-02 | 2017-11-24 | 广州准优生物科技有限公司 | Intacellin and its preparation method and application |
| CN107541486A (en) * | 2017-08-23 | 2018-01-05 | 奥尔文（深圳）生物科技有限公司 | A kind of placenta Subaerial blue green algae active factors of beauty and skin care and its preparation and application |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111588737A (en) * | 2020-04-17 | 2020-08-28 | 焕生汇生物基因技术(北京)有限公司 | Efficient preparation method of placenta extract containing multiple growth factors |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108186548A (en) | A kind of preparation method of the stem cell factor Essence with anti-aging effects | |
| CN102552099B (en) | Composite biological agent used for skin beautifying and restoring and preparing method | |
| CN106519020B (en) | A kind of functional peptide and its preparation method and application with beauty functions | |
| JP2001163899A (en) | Method for producing functional silk fibroin and its use | |
| JP2002128691A (en) | Sericin-containing raw material, method for producing the same and method for using the same | |
| CN105176914A (en) | Simultaneous autologous epidermic cell and fibrocyte preparation method and biological cosmetic product thereof | |
| CN109207545B (en) | Collagen extraction method | |
| CN104548209B (en) | Tissue-engineered epidermis and preparation method thereof | |
| CN101802177A (en) | Matter stem cell and extract the method for its secretory product between extracting from the human or animal embryo | |
| CN103505377A (en) | Polypeptide composition with skin activity | |
| CN104195205A (en) | Method for preparing high-activity collagen peptide from animal placentae and prepared high-activity collagen peptide and application of high-activity collagen peptide | |
| CN103911383B (en) | Be suitable for transformation human acid fibroblast growth factor gene that domestic natural silk gland expresses and expression system and application | |
| CN104814980A (en) | Production method and applications of human embryo fibroblasts | |
| CN110538312A (en) | skin wound repair ointment and preparation method thereof | |
| CN107349418A (en) | A kind of basic fibroblast growth factor composition and preparation method thereof, preparation for baldness and preparation method thereof and application method | |
| CN104173252B (en) | A kind of biological beauty preparation of the cell containing autologous substrate | |
| CN100402548C (en) | Method for preparing physiological active polypeptide of deer placenta | |
| CN105056307A (en) | Artificial skin and preparation method thereof | |
| CN112957286A (en) | Recombinant keratin medical mask and preparation method thereof | |
| CN104611289A (en) | Method for simultaneously preparing autologous epidermal cells and fibroblasts, and biological beauty product comprising autologous epidermal cells and fibroblasts | |
| CN110292628A (en) | Hair follicle stem cells and fat stem cell composite growth factor preparation method and application | |
| CN107683148B (en) | Skin reconstruction method | |
| CN108578264A (en) | Placental hormone and its preparation, application | |
| KR102130988B1 (en) | manufacturing method of the complex of Honey, Wax gourd, Trichosanthis, Omija, Ginseng, Mulberry tree, Pagoda tree fermented low temperature and cosmetic composition for anti-aging comprising the same | |
| CN105853341B (en) | A kind of anti-aging skin care product and preparation method thereof containing epidermal stem cells secretin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180928 |