CN104498425B - The isolation cultivation method of saussurea involucrata stem cell and preparing the application in anti-aging product - Google Patents
The isolation cultivation method of saussurea involucrata stem cell and preparing the application in anti-aging product Download PDFInfo
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- CN104498425B CN104498425B CN201410715007.1A CN201410715007A CN104498425B CN 104498425 B CN104498425 B CN 104498425B CN 201410715007 A CN201410715007 A CN 201410715007A CN 104498425 B CN104498425 B CN 104498425B
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- 238000002955 isolation Methods 0.000 title abstract description 9
- 238000012364 cultivation method Methods 0.000 title abstract description 8
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Abstract
The invention discloses the isolation cultivation method of a kind of saussurea involucrata stem cell and preparing the application in anti-aging product.Specifically get Herba Saussureae Involueratae young leaflet tablet, first utilize solid medium to be separated Herba Saussureae Involueratae stem cell to after its sterilization, recycling liquid nutrient medium to its go down to posterity join foster; Described solid medium contains inorganic salt, VITAMIN, amino acid, growth hormone, carbohydrate and gac; Liquid nutrient medium contains inorganic salt, VITAMIN, amino acid, growth hormone, carbohydrate and additive.Human adipose mesenchymal stem cells extract is improve the activity of SOD enzyme in cell; thus reduce because the oxyradical of ultraviolet radiation induction generation is to the attack of cell and infringement; reduce the mortality ratio of cell, thus play the effect of Cell protection, reach effect of anti-light aging.
Description
Technical field
The present invention relates to the application of plant stem cell at anti-aging product, particularly relate to the isolation cultivation method of saussurea involucrata stem cell and preparing the application in anti-aging product.
Background technology
Stem cell is a kind of seed cell of not yet differentiation and development, has unlimited self and copies and differentiation potential, and this is the basis of stem cell for cosmetics.Stem cell beautifying technique and related products thereof are subject to the extensive concern of beauty treatment circle expert and insider for nearly 2 years.All stem cells, no matter their source (plant, animal or the mankind) are all containing the special epigenetic factor and abundant nutritive substance, these materials are the self-renewal capacities maintaining stem cell.
Plant stem cell technology is based on the wound recovery mechanisms of plant, Schilling plant forms wound thus histiocytic formation is restored in induction, restore tissue and contain undifferentiated cell and stem cell, these recoveries are organized cell harvesting to get up and cultivated in nutrient solution, contain fructose, trace element and plant hormone in nutrient solution, be then positioned over bio-reaction system and carry out scale operation.
Saussurea involucrata is composite family hair phoenix Chrysanthemum succulent, is composite family per nnial herb.Containing abundant cell in saussurea involucrata, especially stem cell.In addition, because it is raw in the mountain in height above sea level 5000m, there air rarefaction will endure the strongest ultraviolet and the invasion and attack of radiation to the fullest extent simultaneously, and therefore its stem cell fundamentally can solve the ubiquitous old and feeble problem of the mankind.
Correlation report and patent is had to claim, animals and plants Stem Cell Activity composition is added in makeup and can play certain anti-wrinkle effect, but the actives really deriving from animals and plants stem cell causes cost of material very expensive because productive rate is low, thus limits its use.In addition, on market today, stem cell products comes from animality Placenta extract or animality stem cell more, and they and human body cell composition are close, thus may be used for anti-ageing effect of waiting for a long time.Then, there is dispute in many countries in the use of animal derived stem cell, not only there is potential safety hazard, have the restriction of many human aspects simultaneously.And plant stem cell Soviet Union good day mostly has totipotency, possess the feature of stem cell, but its homology with human body is more, therefore the function that possesses of plant stem cell extract is current is not also fully aware of, hinders corresponding application.
Summary of the invention
Current biffins cell by first Application in makeup, and the research of saussurea involucrata stem cell not to be reported at present, so the present invention restores the plant stem cell of the higher physiologically active of extraction and isolation tissue first from saussurea involucrata, and carries out scale operation; Be made into lyophilized powder, be added in certain proportion in anti-aging beauty-care product.
The object of the invention is achieved through the following technical solutions:
An isolation cultivation method for saussurea involucrata stem cell, is characterized in that, gets Herba Saussureae Involueratae young leaflet tablet, first utilizes solid medium to be separated Herba Saussureae Involueratae stem cell to after its sterilization, recycling liquid nutrient medium to its go down to posterity join foster; Described solid medium contains inorganic salt, VITAMIN, amino acid, growth hormone, carbohydrate and gac; Liquid nutrient medium contains inorganic salt, VITAMIN, amino acid, growth hormone, carbohydrate and additive.
Preferably include following steps:
(1) saussurea involucrata young leaflet tablet is got, cleaning, sterilization;
(2) by sterilization after saussurea involucrata blade inoculation on solid medium; Described solid medium contains inorganic salt, VITAMIN, amino acid, growth hormone, carbohydrate, gac and agar; Culture temperature is 25-29 DEG C, and pH value is 6.0-6.5, and incubation time is 20-25 days, obtains deriving from cambial saussurea involucrata stem cell;
(3) will derive from cambial saussurea involucrata stem cell to be transferred in liquid culture medium and to cultivate, described liquid nutrient medium contains inorganic salt, VITAMIN, amino acid, growth hormone, carbohydrate and additive.
Before step (2) described inoculation, first saussurea involucrata young leaflet tablet after sterilization is cut into about 0.5cm*0.5cm size, during inoculation, leaf back is contacted with substratum, 1 every bottle;
Step (2) described inorganic salt are more than one in saltpetre, magnesium sulfate, manganous sulfate, zinc sulfate, copper sulfate, calcium chloride, potassiumiodide, cobalt chloride, SODIUM PHOSPHATE, MONOBASIC or boric acid; The content of each material is: saltpetre 2450-2500mg/L, magnesium sulfate 122-124mg/L, manganous sulfate 11-13mg/L, zinc sulfate 2.0-3.0mg/L, copper sulfate 0.03-0.05mg/L, calcium chloride 112-114mg/L, potassiumiodide 0.7-0.75mg/L, cobalt chloride 0.024-0.026mg/L, SODIUM PHOSPHATE, MONOBASIC 132-135mg/L, boric acid 2.8-3.0mg/L;
VITAMIN is more than one in inositol, thiamines class, nicotinic acid, L-AA or citric acid; The content of each material is: inositol 210-230mg/L, thiamines class 19-21mg/L, nicotinic acid 1.6-2.2mg/L, L-AA 52-55mg/L, citric acid 75-85mg/L;
Amino acid is more than one in L-Aspartic acid, L arginine, glycine or proline(Pro); The content of each material is: L-Aspartic acid 135-145mg/L, L arginine 160-180mg/L, glycine 78-85mg/L, proline(Pro) 118-126mg/L;
Growth hormone is α-naphthaleneacetic acid, and content is 1.8-3.0mg/L;
Carbohydrate is sucrose or fructose, and content is 10.0-12.0mg/L;
The content of gac is 450-550mg/L;
The content of agar is 3.6-3.8mg/L;
Step (3) described inorganic salt are more than one in saltpetre, magnesium sulfate, manganous sulfate, zinc sulfate, copper sulfate, calcium chloride, potassiumiodide, cobalt chloride, SODIUM PHOSPHATE, MONOBASIC or boric acid;
The content of each material is: saltpetre 992-996mg/L, magnesium sulfate 180-184mg/L, manganous sulfate 22-26mg/L, zinc sulfate 10-12mg/L, copper sulfate 0.3-0.5mg/L, calcium chloride 72-76mg/L, potassiumiodide 0.3-0.6mg/L, cobalt chloride 0.03-0.036mg/L, SODIUM PHOSPHATE, MONOBASIC 126-131mg/L, boric acid 2.0-2.5mg/L;
VITAMIN is more than one in inositol, thiamines class, nicotinic acid, L-AA or citric acid; Each substances content is: inositol 194-220mg/L, thiamines class 20-23mg/L, nicotinic acid 3-5mg/L, L-AA 57-62mg/L, citric acid 75-83mg/L;
Amino acid is more than one in L-Aspartic acid, L arginine, glycine or proline(Pro); The content of each material is: L-Aspartic acid 136-142mg/L, L arginine 172-180mg/L, glycine 75.0-85.0mg/L, proline(Pro) 110.0-115.0mg/L;
Growth hormone is α-naphthaleneacetic acid, and content is 3.0-5.0mg/L;
Carbohydrate is sucrose or fructose, and content is 32.0-34.0mg/L;
Additive is medical stone granule: and selenium ore particles, medical stone granule: 60.0-80.0mg/L, selenium ore particles 60.0-80.0mg/L.
Present invention also offers a kind of method utilizing described isolation cultivation method to prepare saussurea involucrata stem cell extract, comprise the following steps:
(1) by the saussurea involucrata stem cell amplification culture of above-mentioned isolation cultivation method acquisition;
(2) broth filtrate, takes out the cloned culture that retains, after freezing, fragmentation, filters, filtrate is concentrated and obtain saussurea involucrata stem cell extract.
Described amplification culture is moved in bio-reactor by the stem cell cultivated in liquid medium within to cultivate, nutrient solution uses liquid culture medium (composition is with the liquid nutrient medium in isolation cultivation method step (3)), uninterruptedly stir at 25 DEG C, when being cultured to 10d, in bio-reactor, inject the mixed solution of sucrose, methyl jasmonate and aseptic magnetized water;
Step (2) described freezing be put into-80 DEG C of superfreeze 2h, take out and to thaw 30min; Put into-80 DEG C of superfreeze again, 3-5 time successively; Utilize sonicator smudge cells after thawing for the last time, metre filter, collect filtrate, concentrated in rotating vacuum evaporator, be saussurea involucrata stem cell extract.
Also comprise after being mixed with sweet dew alcohol and water by saussurea involucrata stem cell extract, degerming, freeze-drying obtains the step of saussurea involucrata stem cell extract freeze-drying powder.Described water is the ultrapure water of 50 ~ 60 DEG C, adopts BCA method to carry out determination of protein concentration to described saussurea involucrata stem cell extract, and according to this protein concentration, adding water, to be adjusted to final concentration of protein be 50.0 ± 0.5 μ g/ml; The quality of described N.F,USP MANNITOL accounts for 5% of saussurea involucrata stem cell extract total mass; The temperature of described freeze-drying is-20 ~-35 DEG C, and vacuum tightness is 50 ~ 200Pa, and freeze-drying time is 48 hours.
The invention provides a kind of saussurea involucrata stem cell extract freeze-drying powder and comprise saussurea involucrata stem cell extract and N.F,USP MANNITOL, wherein the quality of N.F,USP MANNITOL accounts for 5% of saussurea involucrata stem cell extract total mass.
The preparation method that present invention also offers described saussurea involucrata stem cell extract obtains saussurea involucrata stem cell extract or the application of saussurea involucrata stem cell extract freeze-drying powder in anti-aging product.
Preferably, described method obtains the obtained a kind of eye cream of saussurea involucrata stem cell extract or saussurea involucrata stem cell extract freeze-drying powder, comprise saussurea involucrata stem cell lyophilized powder 0.1-20ng, Firm agent 2.0-3.5g, remove wrinkle agent 0.5-2.5g, acrylate copolymer 0.5-2.5g, emulsifying agent 1.0-3.5g, Wickenol 111 3.5-5.5g and synthesis squalane 0.5-3.5g.
The preparation technology of eye cream of the present invention comprises the steps:
By acrylate copolymer deionized water heating for dissolving, stir, component A is in 70 ~ 80 DEG C of dissolvings, component A is poured in acrylate copolymer, stir, component C and D component is added after being cooled to 37 ~ 50 DEG C, stir, then prepare the saussurea involucrata stem cell lyophilized powder eye cream of the present embodiment.Component A comprises emulsifying agent, Wickenol 111 and synthesis squalane.Component C comprises Firm agent and goes agent of wrinkling, and D component is the saussurea involucrata stem cell lyophilized powder after dissolving with stablizer.
The advantage that the present invention has relative to prior art and beneficial effect:
Human adipose mesenchymal stem cells extract is improve the activity of SOD enzyme in cell; thus reduce because the oxyradical of ultraviolet radiation induction generation is to the attack of cell and infringement; reduce the mortality ratio of cell, thus play the effect of Cell protection, reach effect of anti-light aging.
Accompanying drawing explanation
Fig. 1 is that saussurea involucrata stem cell lyophilized powder is on the impact of SOD enzyme activity;
Fig. 2 is that saussurea involucrata stem cell lyophilized powder is on SOD enzyme activity impact in mice plasma;
Fig. 3 is that saussurea involucrata stem cell lyophilized powder is to GPx activity influence in mice plasma;
Fig. 4 is that saussurea involucrata stem cell lyophilized powder is to MDA activity influence in mice plasma.
Embodiment
Embodiment 1
(1) preparation of Herba Saussureae Involueratae material
The present invention gets Herba Saussureae Involueratae young leaflet tablet, blade tap water running water 2h; With concentration 70% alcohol surface sterilization 10s, aseptic water washing 3 times; With concentration 0.1% liter of tribute solution sterilize 4 respectively, 6,8, after 10min, with aseptic water washing 3 times, what wash away plant surface rises tribute raffinate, to form different disinfecting; Pasteurization material is put into sterile petri dish, for subsequent use with aseptic filter paper suck dry moisture.
(2) from material, Herba Saussureae Involueratae stem cell is separated
Under aseptic condition, saussurea involucrata young leaflet tablet after sterilization is cut into about 0.5cm*0.5cm size, is seeded on solid medium.During inoculation, leaf back is contacted with substratum, 1 piece every bottle.Culture temperature is 25 DEG C, and cultivate pH value 6.0, incubation time is 20 days.
Solid culture based component is saltpetre 2450mg/L; Inositol 210mg/L; L-Aspartic acid 135mg/L; α-naphthaleneacetic acid 1.8mg/L, sucrose 10.0mg/L, gac 450mg/L, agar 3.6mg/L, with 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 98kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 20min saves backup.
(3) Secondary Culture
In time being cultured to 20 days, cambial cell being transferred in the flask preferably comprising liquid culture medium and cultivating, and the moment examines under a microscope state of aggregation and the degree of cell.
Liquid nutrient medium is: saltpetre 992mg/L, nicotinic acid 3.0mg/L, glycine 75mg/L, α-naphthaleneacetic acid 3.0mg/L, sucrose 32.0mg/L, medical stone granule: 60.0mg/L and selenium ore particles 60.0g/L, all the other are water.With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 98kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 20min saves backup.
(4) Bioreactor scaleup is cultivated
Amplification culture in the bio-reactor stem cell cultivated in flask being moved into 20L, nutrient solution uses preferred liquid culture medium in (3), uninterruptedly stirs at 25 DEG C.When being cultured to 10d, in bio-reactor, injecting the mixed solution of sucrose, methyl jasmonate and aseptic magnetized water, accelerating stem cell growth with supplementary later stage nutrition.
(5) cell harvesting and fragmentation
Nutrient solution is used 1um metre filter, remove nutrient solution, the cloned culture retained is taken out, puts into-80 DEG C of super low temperature quick frozen 2h, take out the 30min that thaws; Put into-80 DEG C of super low temperature quick frozens again, 3 times successively; Utilize sonicator smudge cells after thawing for the last time, metre filter, collect filtrate, concentrated in rotating vacuum evaporator, be saussurea involucrata stem cell extract solution.
(6) lyophilized powder preparation
BCA method is adopted to carry out determination of protein concentration to saussurea involucrata stem cell extract solution, according to this protein concentration, add the ultrapure water mixing of vehicle N.F,USP MANNITOL (N.F,USP MANNITOL accounts for 5% of saussurea involucrata stem cell extract solution gross weight) and 50 DEG C, and to be adjusted to final concentration of protein be 50.0+0.5 μ g/ml, filtration sterilization, then carries out frozen dried, freeze temperature is-20 DEG C, vacuum tightness is 50Pa, and freeze-drying time is 48 hours, then prepare required saussurea involucrata stem cell extract freeze-drying powder; Lyophilized powder according to 1ml measure/prop up packing.
Embodiment 2
(1) preparation of Herba Saussureae Involueratae material
The present invention gets Herba Saussureae Involueratae young leaflet tablet, blade tap water running water 4h; With concentration 75% alcohol surface sterilization 15s, aseptic water washing 5 times; With concentration 0.1% liter of tribute solution sterilize 4 respectively, 6,8, after 10min, with aseptic water washing 5 times, what wash away plant surface rises tribute raffinate, to form different disinfecting; Pasteurization material is put into sterile petri dish, for subsequent use with aseptic filter paper suck dry moisture.
(2) from material, Herba Saussureae Involueratae stem cell is separated
Under aseptic condition, saussurea involucrata young leaflet tablet after sterilization is cut into about 0.5cm*0.5cm size, is seeded on solid medium.During inoculation, leaf back is contacted with substratum, 1 piece every bottle.Culture temperature is 29 DEG C, and cultivate pH 6.5, incubation time is about 30 days.
Solid medium is: cobalt chloride 0.025mg/l, L-AA 54mg/l; Proline(Pro) 120mg/l; Growth hormone is α-naphthaleneacetic acid 2.5mg/l, carbohydrate is sucrose 11.0mg/l, additive is gac 500mg/L, agar 3.7mg/l.With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 108kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 25min saves backup.
(3) Secondary Culture
In time being cultured to 30 days, cambial cell being transferred in the flask preferably comprising liquid culture medium and cultivating, and the moment examines under a microscope state of aggregation and the degree of cell.
The composition of liquid nutrient medium is: SODIUM PHOSPHATE, MONOBASIC 130mg/l; Citric acid 80mg/l; Proline(Pro) 114.0mg/l; Growth hormone is α-naphthaleneacetic acid 4.0mg/l, carbohydrate is sucrose 33.0mg/l, additive is medical stone granule: 70.0mg/l and selenium ore particles 70.0mg/l, and all the other are water.With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 108kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 25min saves backup.
(4) Bioreactor scaleup is cultivated
Amplification culture in the bio-reactor stem cell cultivated in flask being moved into 20L, nutrient solution uses preferred liquid culture medium in (3), uninterruptedly stirs at 25 DEG C.When being cultured to 10d, in bio-reactor, injecting the mixed solution of sucrose, methyl jasmonate and aseptic magnetized water, accelerating stem cell growth with supplementary later stage nutrition.
(5) cell harvesting and fragmentation
Nutrient solution is used 1um metre filter, remove nutrient solution, the cloned culture retained is taken out, puts into-80 DEG C of super low temperature quick frozen 2h, take out the 30min that thaws; Put into-80 DEG C of super low temperature quick frozens again, 5 times successively; Utilize sonicator smudge cells after thawing for the last time, metre filter, collect filtrate, concentrated in rotating vacuum evaporator, be saussurea involucrata stem cell extract solution.
(6) lyophilized powder preparation
BCA method is adopted to carry out determination of protein concentration to saussurea involucrata stem cell extract solution, according to this protein concentration, add the ultrapure water mixing of vehicle N.F,USP MANNITOL (N.F,USP MANNITOL accounts for 5% of saussurea involucrata stem cell extract solution gross weight) and 60 DEG C, and to be adjusted to final concentration of protein be 49.5 μ g/ml, filtration sterilization, then carries out frozen dried, freeze temperature is-35 DEG C, vacuum tightness is 200Pa, and freeze-drying time is 48 hours, then prepare required saussurea involucrata stem cell extract freeze-drying powder; Lyophilized powder according to 1ml measure/prop up packing.
Embodiment 3
(1) preparation of Herba Saussureae Involueratae material
The present invention gets Herba Saussureae Involueratae young leaflet tablet, blade tap water running water 3h; With concentration 75% alcohol surface sterilization 15s, aseptic water washing 4 times; With concentration 0.1% liter of tribute solution sterilize 4 respectively, 6,8, after 10min, with aseptic water washing 4 times, what wash away plant surface rises tribute raffinate, to form different disinfecting; Pasteurization material is put into sterile petri dish, for subsequent use with aseptic filter paper suck dry moisture.
(2) from material, Herba Saussureae Involueratae stem cell is separated
Under aseptic condition, saussurea involucrata young leaflet tablet after sterilization is cut into about 0.5cm*0.5cm size, is seeded on solid medium.During inoculation, leaf back is contacted with substratum, 1 piece every bottle.Culture temperature is 27 DEG C, and cultivate pH value 6.0, incubation time is 15 days.
Solid culture based component is: potassiumiodide 0.75mg/l, cobalt chloride 0.026mg/l; Nicotinic acid 2.2mg/l, glycine 85mg/l; Growth hormone is α-naphthaleneacetic acid 3.0mg/l, carbohydrate is fructose 12.0mg/ml, additive is gac 500.0mg/l, agar 3.8mg/l; With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 100kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 15min saves backup.
(3) Secondary Culture
In time being cultured to 15 days, cambial cell being transferred in the flask preferably comprising liquid culture medium and cultivating, and the moment examines under a microscope state of aggregation and the degree of cell.
The composition of liquid nutrient medium is: SODIUM PHOSPHATE, MONOBASIC 131mg/l, boric acid 2.5mg/l; L-AA 62.0mg/l, citric acid 83.0mg/l; L-Aspartic acid 142.0mg/l, L arginine 180mg/l; Growth hormone is α-naphthaleneacetic acid 5.0mg/l, carbohydrate is sucrose 34.0mg/l, additive is medical stone granule: 80mg/l and selenium ore particles 80mg/l, and all the other are water.With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 100kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 15min saves backup.
(4) Bioreactor scaleup is cultivated
Amplification culture in the bio-reactor stem cell cultivated in flask being moved into 20L, nutrient solution uses preferred liquid culture medium in (3), uninterruptedly stirs at 25 DEG C.When being cultured to 10d, in bio-reactor, injecting the mixed solution of sucrose, methyl jasmonate and aseptic magnetized water, accelerating stem cell growth with supplementary later stage nutrition.
(5) cell harvesting and fragmentation
Nutrient solution is used 1um metre filter, remove nutrient solution, the cloned culture retained is taken out, puts into-80 DEG C of super low temperature quick frozen 2h, take out the 30min that thaws; Put into-80 DEG C of super low temperature quick frozens again, 4 times successively; Utilize sonicator smudge cells after thawing for the last time, metre filter, collect filtrate, concentrated in rotating vacuum evaporator, be saussurea involucrata stem cell extract solution.
(6) lyophilized powder preparation
BCA method is adopted to carry out determination of protein concentration to saussurea involucrata stem cell extract solution, according to this protein concentration, add the ultrapure water mixing of vehicle N.F,USP MANNITOL (N.F,USP MANNITOL accounts for 5% of saussurea involucrata stem cell extract solution gross weight) and 55 DEG C, and to be adjusted to final concentration of protein be 50.0 μ g/ml, filtration sterilization, then carries out frozen dried, freeze temperature is-30 DEG C, vacuum tightness is 100Pa, and freeze-drying time is 48 hours, then prepare required saussurea involucrata stem cell extract freeze-drying powder; Lyophilized powder according to 1ml measure/prop up packing.
Saussurea involucrata stem cell lyophilized powder eye cream
Eye cream composition divides into groups by the present embodiment, as shown in table 1, prepares the eye cream of the saussurea involucrata stem cell lyophilized powder containing preparation of 3 kinds of formulas.
Table 1
The preparation technology of the present embodiment eye cream comprises the steps:
By B component portions of de-ionized water heating for dissolving, stir, component A is in 70 DEG C of dissolvings, and component A is poured in B component, stir, add component C and D component after being cooled to 37 DEG C, stir, then prepare the saussurea involucrata stem cell lyophilized powder eye cream of the present embodiment.
The photoaging of saussurea involucrata stem cell lyophilized powder In Vitro Anti is tested
Detect the obtained saussurea involucrata stem cell lyophilized powder of embodiment 1 to the provide protection of ultraviolet damage fibroblasts of adult human dermis
Detect sample: saussurea involucrata stem cell lyophilized powder prepared by embodiment 1.
Biological subject body: HDF cell.
Concrete steps are as follows:
(1) when fibroblasts of adult human dermis grows to 80% fusion, with UVA uviolizing cell, irradiate 2 hours every day, concurrent irradiation 4 days, normal group tinfoil encases Tissue Culture Flask.Examine under a microscope the form of cell, find that cell starts to occur shrinkage, respective cells is in the medium floating; 5th day, add saussurea involucrata stem cell lyophilized powder and continue to cultivate (add rear final concentration of protein and be respectively 20 μ g/ml and 10 μ g/ml), Catergen 0 μ g/ml, as positive control, continues to cultivate 48h, collecting cell;
(2) saussurea involucrata stem cell lyophilized powder is on the impact of SOD enzyme activity in cell: after trysinization, and cell is centrifugal 10min under 1500g, 4 DEG C of conditions.Cell precipitation is suspended in (Tris – HCl, 5mMEDTA and 1mMDTT of 50mM) in the cold damping fluid of chance and carries out ultrasonic, lysate is centrifugal 10min under 10000g, 4 DEG C of conditions, and supernatant utilizes SOD detection kit to analyze according to its illustration method.
(3) result is illustrated in fig. 1 shown below: no matter human adipose mesenchymal stem cells extract is 20 μ g/ml; or 10 μ g/ml are in the activity that improve SOD enzyme in cell in varying degrees; thus reduce because the oxyradical of ultraviolet radiation induction generation is to the attack of cell and infringement; reduce the mortality ratio of cell; thus play the effect of Cell protection, reach effect of anti-light aging.
In figure, 1 is saussurea involucrata stem cell extract freeze-drying powder 20 μ g/ml group; 2 is saussurea involucrata stem cell extract freeze-drying powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is normal group; Wherein: * * P≤0.01, * P≤0.05
Saussurea involucrata stem cell lyophilized powder causes the provide protection of Mice Photoaging to ultraviolet
1. detect sample: the saussurea involucrata stem cell lyophilized powder (protein content is that 50.0 ± 0.5 μ g/ prop up) of preparation.
2. test method:
2.1 animal rearings: in SPF level female KM mouse in 4 week age (body weight 20 ± 2g), Guangdong Province's Medical experimental center provides, after adaptability raises one week, reject mouse back hair, baring skin cotton knot is about 2cm2, tests.Normal feedwater and feed in process of the test.
2.2 experiment grouping and drug treating: mouse is divided into five groups, often organizes 5, proceeds as follows respectively:
1st group: the saussurea involucrata stem cell lyophilized powder prepared with physiological saline solution, the saussurea involucrata stem cell lyophilized powder concentration preparing preparation is the solution of 20 μ g/ml; Be applied on mouse, applying amount is lml/, 1 times/day of (pre-irradiation), then uv irradiating;
2nd group: the saussurea involucrata stem cell lyophilized powder prepared with physiological saline solution, the saussurea involucrata stem cell lyophilized powder concentration of preparation preparation is the solution of 10 μ g/ml; ; Be applied on mouse, applying amount is lml/, 1 times/day of (pre-irradiation), then uv irradiating;
3rd group: model group, the mouse of this group does not smear saussurea involucrata stem cell lyophilized powder, uv irradiating;
4th group: positive controls, prepare with physiological saline solution vitamins C (VitC) solution that vitamine concentration is 20 μ g/ml, be applied on mouse by this solution, applying amount is lml/, 1 times/day of (pre-irradiation), then uv irradiating; 5th group: normal group, the mouse of this group is not smeared whatever, also not uv irradiating.
The concrete operations of uviolizing: after lyophilized powder is absorbed, uv irradiating is carried out to mouse administration 1h, first week every day irradiated 1h, and second week irradiates 2h every day, and the 3rd week every day irradiated 3h, and from the 4th week, every day irradiated 4h, total concurrent irradiation 42 days; Ultraviolet intensity is: UVA:0.25mJ/cm2/s, UVB:0.02mJ/cm2/s; To be illuminated complete after, by mouse put to death, carry out the detection of indices; Carry out bloodletting to mouse, serum is centrifugal 4min under 2500g, 4 DEG C of conditions, gets the detection that supernatant carries out indices.
3. test item:
The mensuration of SOD enzyme, Selenoperoxidase (GPx) and mda (MDA) content in blood plasma: bloodletting is carried out to mouse, centrifugal 4min under 2500g, 4 DEG C of conditions, collects supernatant;
The activity of SOD, the diagnostic kit that the activity of GPx and the content of MDA utilize Nanjing to build up Bioengineering Research Institute to be provided carries out determination and analysis according to the specification sheets of supplier.
Skin slice HE dyes, and observe the change of skin texture: the skin getting mouse back outshot wool, the formaldehyde solution of 10% is fixed, and routine paraffin wax is cut into slices, and HE dyes observation.
4. detected result:
1) following Fig. 2 of the detected result of SOD enzyme activity in mice plasma; as seen from the figure; although SOD enzyme activity does not return to levels found in normal controls in the Mice Body of saussurea involucrata stem cell lyophilized powder protection; but compare with model group and positive drug VitC group; equal tool is greatly improved; illustrate that the objectionable impuritiess such as the oxyradical in animal body in antioxidant system purged body are enough more effectively removed or assisted to saussurea involucrata stem cell lyophilized powder, thus make anti-oxidant equilibrium system keep stable.
In figure, 1 is saussurea involucrata stem cell lyophilized powder 20 μ g/ml group; 2 is saussurea involucrata stem cell lyophilized powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is normal group; Wherein: * * P≤0.01, * P≤0.05
2) following Fig. 3 of the detected result of GPx activity in mice plasma, as seen from Figure 3, the GPx activity of the 1st group of mouse and the 2nd group of mouse all than model group and positive drug VitC group good, thus protect mouse skin.
In Fig. 3,1 is saussurea involucrata stem cell lyophilized powder 20 μ g/ml group; 2 is saussurea involucrata stem cell lyophilized powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is normal group; Wherein: * * P≤0.01, * P≤0.05
3) following Fig. 4 of the detected result of MDA content in mice plasma, as seen from the figure, human adipose mesenchymal stem cells extract significantly reduces the content of MDA, maintains anti-oxidant equilibrium system in body.
In figure, 1 is saussurea involucrata stem cell lyophilized powder 20 μ g/ml group; 2 is saussurea involucrata stem cell lyophilized powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is normal group; Wherein: * * P≤0.01, * P≤0.05
4) detected result of the mice skin tissue of 5 experimental group is as follows:
1st group: skin texture is complete and clear, and normal skin tissue does not have obvious difference, has no the phenomenons such as inflammatory cell infiltration;
2nd group: intradermal arrangement of collagen fibers is comparatively neat, and the appendages of skin have a little hyperplasia;
3rd group (model group): mouse skin thickness obviously increases, and dermis thickness sharply reduces, collegen filament abnormal growth and arrangement disorder, the appendages of skin present skin initial stage aging feature;
4th group (positive controls): epidermis has certain hyperplasia phenomenon, and deep skin arrangement of collagen fibers is comparatively loose;
5th group (normal group): skin is normal, does not have obvious slight crack and hyperplasia phenomenon, does not have the feature that the initial stage is aging.
Due to the medicine with good sun-proof result that vitamins C is known, therefore as the positive drug of this experiment, and net result shows that the various dose group of saussurea involucrata stem cell lyophilized powder has anti-ageing or repair preferably, and effect is equal to positive controls.
The announcement of book and instruction according to the above description, those skilled in the art in the invention can also change above-mentioned embodiment and revise.Therefore, the present invention is not limited to embodiment disclosed and described above, also should fall in the protection domain of claim of the present invention modifications and changes more of the present invention.In addition, although employ some specific terms in this specification sheets, these terms just for convenience of description, do not form any restriction to the present invention.
Claims (1)
1. an antidotal eye cream, is characterized in that, its preparation method comprises the steps:
Step 1: the preparation of Herba Saussureae Involueratae material
Get Herba Saussureae Involueratae young leaflet tablet, blade tap water running water 3h; With concentration 75% alcohol surface sterilization 15s, aseptic water washing 4 times; With concentration 0.1% liter of tribute solution sterilize 4 respectively, 6,8, after 10min, with aseptic water washing 4 times, what wash away plant surface rises tribute raffinate, to form different disinfecting; Pasteurization material is put into sterile petri dish, for subsequent use with aseptic filter paper suck dry moisture;
Step 2: be separated Herba Saussureae Involueratae stem cell from material
Under aseptic condition, saussurea involucrata young leaflet tablet after sterilization is cut into about 0.5cm*0.5cm size, is seeded on solid medium; During inoculation, leaf back is contacted with substratum, 1 piece every bottle; Culture temperature is 27 DEG C, and cultivate pH value 6.0, incubation time is 15 days;
Solid culture based component is: potassiumiodide 0.75mg/l, cobalt chloride 0.026mg/l; Nicotinic acid 2.2mg/l, glycine 85mg/l; Growth hormone is α-naphthaleneacetic acid 3.0mg/l, carbohydrate is fructose 12.0mg/ml, additive is gac 500.0mg/l, agar 3.8mg/l; With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 100kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 15min saves backup;
Step 3: Secondary Culture
In time being cultured to 15 days, cambial cell being transferred in the flask preferably comprising liquid culture medium and cultivating, and the moment examines under a microscope state of aggregation and the degree of cell;
The composition of liquid nutrient medium is: SODIUM PHOSPHATE, MONOBASIC 131mg/l, boric acid 2.5mg/l; L-AA 62.0mg/l, citric acid 83.0mg/l; L-Aspartic acid 142.0mg/l, L arginine 180mg/l; Growth hormone is α-naphthaleneacetic acid 5.0mg/l, carbohydrate is sucrose 34.0mg/l, additive is medical stone granule: 80mg/l and selenium ore particles 80mg/l, and all the other are water; With 0.1%NaOH or HCl adjust pH to 5.8, under the condition of temperature 121 DEG C, pressure 100kPa, the rearmounted sterilisable chamber of high pressure steam sterilization 15min saves backup;
Step 4: Bioreactor scaleup is cultivated
Amplification culture in the bio-reactor stem cell cultivated in flask being moved into 20L, nutrient solution uses the liquid culture medium in step 3, uninterruptedly stirs at 25 DEG C; When being cultured to 10d, in bio-reactor, injecting the mixed solution of sucrose, methyl jasmonate and aseptic magnetized water, accelerating stem cell growth with supplementary later stage nutrition;
Step 5: cell harvesting and fragmentation
By nutrient solution 1 μm of metre filter, remove nutrient solution, the cloned culture retained is taken out, puts into-80 DEG C of super low temperature quick frozen 2h, take out the 30min that thaws; Put into-80 DEG C of super low temperature quick frozens again, 4 times successively; Utilize sonicator smudge cells after thawing for the last time, metre filter, collect filtrate, concentrated in rotating vacuum evaporator, be saussurea involucrata stem cell extract solution;
Step 6: prepared by lyophilized powder
Adopt BCA method to carry out determination of protein concentration to saussurea involucrata stem cell extract solution, according to this protein concentration, add the ultrapure water mixing of vehicle N.F,USP MANNITOL and 55 DEG C, make N.F,USP MANNITOL account for 5% of saussurea involucrata stem cell extract solution gross weight; And to be adjusted to final concentration of protein be 50.0 μ g/ml, filtration sterilization, then carries out frozen dried, and freeze temperature is-30 DEG C, and vacuum tightness is 100Pa, and freeze-drying time is 48 hours, then prepare required saussurea involucrata stem cell extract freeze-drying powder; Lyophilized powder according to 1ml measure/prop up packing;
Saussurea involucrata stem cell lyophilized powder eye cream:
Eye cream composition is divided into groups, prepares the eye cream of the saussurea involucrata stem cell lyophilized powder containing preparation of 3 kinds of formulas:
Formula I comprises component A, B component, component C and D component; Wherein, component A comprises PEA3.5g, IPP5.5g, synthesis squalane 0.5g; B component comprises AVC0.5g; Component C comprises Polylift2.0g, Fermiskin2.3g, extremely beautiful 0.5g, water 79.9g; D component comprises saussurea involucrata stem cell lyophilized powder 0.1ng, stablizer 0.01M phosphate buffered saline buffer 5.0g, and described saussurea involucrata stem cell lyophilized powder adds after first dissolving with stablizer again;
Or formula II comprises component A, B component, component C and D component; Wherein, component A comprises PEA2.0g, IPP4.0g, synthesis squalane 2.0g; B component comprises AVC1.5g; Component C comprises Polylift3.0g, Fermiskin1.5g, extremely beautiful 1.5g, water 78.5g; D component comprises saussurea involucrata stem cell lyophilized powder 10.0ng, stablizer 0.01M phosphate buffered saline buffer 5.0g, and described saussurea involucrata stem cell lyophilized powder adds after first dissolving with stablizer again;
Or formula III comprises component A, B component, component C and D component; Wherein, component A comprises PEA1.0g, IPP3.5g, synthesis squalane 3.5g; B component comprises AVC2.5g; Component C comprises Polylift3.5g, Fermiskin0.5g, extremely beautiful 2.5g, water 76.0g; D component comprises saussurea involucrata stem cell lyophilized powder 20.0ng, stablizer 0.01M phosphate buffered saline buffer 5.0g, and described saussurea involucrata stem cell lyophilized powder adds after first dissolving with stablizer again;
By B component portions of de-ionized water heating for dissolving, stir, component A is in 70 DEG C of dissolvings, and component A is poured in B component, stirs, and adds component C and D component, stir after being cooled to 37 DEG C, then prepare saussurea involucrata stem cell lyophilized powder eye cream.
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