CN109453074A - A kind of double algae compound extracts and its application - Google Patents
A kind of double algae compound extracts and its application Download PDFInfo
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- CN109453074A CN109453074A CN201811374260.XA CN201811374260A CN109453074A CN 109453074 A CN109453074 A CN 109453074A CN 201811374260 A CN201811374260 A CN 201811374260A CN 109453074 A CN109453074 A CN 109453074A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9711—Phaeophycota or Phaeophyta [brown algae], e.g. Fucus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
Abstract
The present invention relates to a kind of double algae compound extracts and its applications.Double algae compound extracts are made of Chlamydomanas nivalis extract and opotism algae extract with mass ratio 1:(1-10).Double algae compound extracts of the present invention can keep good cell viability to recombination epidermal cell, increase the expression of P63 and Klotho；Efficacy assessments the result shows that, the compound extract not only in a short time can General Promotion skin moisture content, brightness, elastic force, skin smoothness, reduce skin surface pigmentation, and subcutaneous Melanin aggregates can be significantly reduced, prevent the formation of color spot.
Description
Technical field
The present invention relates to a kind of with short-term double algae compound extracts of senile-resistant efficacy and its application comprehensively, belongs to skin care
Product technical field.
Background technique
The relevant cosmetic material of existing anti-aging mainly has anti-oxidant class, such as VC, metallothionein from the mechanism of action point
It is white etc.；Stimulation collagen, elastin laminin synthesize class, such as collagen class, peptides；Adjust skin micro ecological, such as lactobacillus-fermented
Product etc.；Anti -inflammatory cytokine class, such as green alga, aloe extract.These raw materials mostly act only on certain spies of skin aging
On targeting point, can not comprehensive function in each target spot of aging, thus in a short time to wrinkle, pigmentation, elastic force, skin water
The effect that part, skin brightness and other effects evaluation index generate comprehensive function is not strong.
Summary of the invention
In order to overcome the above technical problems, the present invention proposes a kind of double algae compound extracts, by selecting specific algae
And take specific extraction process, make to play synergistic effect between opotism algae extract and Chlamydomanas nivalis extract, can it is short-term,
Comprehensively wrinkle, pigmentation, elastic force, skin moisture, in terms of play effect.
The technical solution used in the present invention is as follows.
A kind of double algae compound extracts, by Chlamydomanas nivalis extract and opotism algae extract with mass ratio 1:(1-10) group
At.
Further, the Chlamydomanas nivalis is selected from a kind of unique extremophile microalgae of Switzerland's high mountain, is a kind of single celled algae
Class biology, can keep active vitality in the environment of close to freezing point.
Further, the opotism algae is selected from a kind of big seaweed of France, and spore is contained in root, is responsible for the numerous of opotism algae
It grows, is considered as " lifespring ".Though opotism algae described herein and common thallus laminariae belong to generic biology, it is special
After assigning to fixed output quota its special performance, with Chlamydomanas nivalis compatibility, synergistic effect can be played, senile-resistant efficacy is significantly improved.
Further, the preparation of the Chlamydomanas nivalis extract, comprising: the acquisition of Chlamydomanas nivalis, culture, homogenate, water mention,
Separation.
Wherein, the acquisition selection acquires Chlamydomanas nivalis in -10 DEG C or less in winter and 10 DEG C of spring or more of period
Sample, it is spare after screening.
Wherein, the environment temperature of the culture is -30 DEG C of -30 DEG C of adjustable extents, and ambient humidity is that 10-90%RH is adjustable
Adjusting range；Meanwhile the environment of the culture must also have illumination linear regulating function, LED cold light source is red 620nm, indigo plant
460nm.After the snow algae of acquisition is sterilized, it is placed in the incubator, by adjusting temperature, humidity, illumination, snow algae is cultivated.
Further, the preparation of the opotism algae extract, comprising: selection opotism algae root crushes, water mentions 4-5h, mistake
Filter；Filter residue adds flooding, repetition secondary, merges filtrate three times；Ethyl alcohol is added, rotary evaporation concentrates the filtrate to the three of volume
/ bis-.
In order to guarantee the effect of extract, in the preparation process of the Chlamydomanas nivalis extract and opotism algae extract,
Preparation temperature is controlled at 60 DEG C or less.
The present invention also provides application of the above-mentioned double algae compound extracts in anti-aging product.
The present invention also provides a kind of double algae compositions, contain above-mentioned double algae compound extracts.Preferably, double algae combinations
Object further includes auxiliary material, such as cyclodextrin, silica, diatomite, for wrapping up double algae compound extracts, formation off-white color
The double algae compositions of anti-aging.
It is obtained by the present invention to have the beneficial effect that:
Double algae compound extracts of the present invention can keep good cell viability to recombination epidermal cell, increase P63
With the expression of Klotho, efficacy assessments the result shows that, which not only in a short time can General Promotion skin moisture content, bright
Degree, elastic force, skin smoothness reduce skin surface pigmentation, and subcutaneous Melanin aggregates can be significantly reduced, and prevent
The formation of color spot.In addition, the present invention, which passes through, first separately extracts the processing method compounded again, it can be by targeted activity in different algal species
Object is sufficiently extracted by different technology conditions, is made in final product rich in a plurality of types of active ingredients, gained compound
With higher practical application value.
Detailed description of the invention
Fig. 1 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
Skin whiteness (brightness) L* value variation tendency.
Fig. 2 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
ITA ° of value variation tendency of skin brightness.
Fig. 3 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
The variation tendency of dermal melanin content MI value.
Fig. 4 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
Skin whiteness (brightness) L* value variation tendency.
Fig. 5 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
The variation tendency of skin elasticity R2 value.
Fig. 6 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
The variation tendency of skin elasticity Q1 value.
Fig. 7 is test zone after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
The change rate of skin elasticity Q1 value.
After Fig. 8 is use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment, subject 1
(number 03) image comparison result.
After Fig. 9 is use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment, subject 2
(number 04) image comparison result.
After Figure 10 is use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment, subject 3
(number 05) desalinates microgroove image comparison result.
After Figure 11 is use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment, subject 4
(number 06) desalinates microgroove image comparison result.
Figure 12 is canthus survey after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
Try the variation tendency of region wrinkle area accounting.
Figure 13 is canthus survey after use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of embodiment
Try the change rate of region wrinkle area accounting.
Figure 14 is use anti-aging essence skincare product as made from the double algae compound extracts of 1 gained of various concentration embodiment
Afterwards, epidermal cell vigor is observed.
Figure 15 is p63 expression after the embodiment 1 of different quality percentage concentration handles epidermis.
Figure 16 is Klotho gene expression dose after the embodiment 1 of different quality percentage concentration handles epidermis.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
A kind of preparation of double algae compound extracts of embodiment 1
The present embodiment provides a kind of preparations of double algae compound extracts, comprising:
1. the preparation of the preparation (component 1) of Chlamydomanas nivalis extract
1) it acquires, culture Chlamydomanas nivalis, culture snow algae (500g) is placed in homogenizer, snow algae polishing pulp obtains
To snow algae slurry；
2) purified water is added in snow algae slurry, mass ratio is snow algae slurry: water=1:2；
3) 2h is stirred under room temperature；
4) decompression suction filtration is carried out, filtrate is collected, obtains snow algae water extract (component 1).
2. the preparation of opotism algae extract (component 2)
1) it will be pulverized into powder by the opotism algae standby (500 grams) of freeze-drying, lixiviation apparatus be added and soaks extracting cylinder
In, 2000ml water is added, is heated to 40-45 DEG C, is slowly stirred, low temperature extracts 4 hours；
2) leaching liquor is evacuated in liquid storage cylinder by filter, residue is collected back in extraction cylinder, and 1000ml water is added, adds
Heat is slowly stirred to 40-45 DEG C, and low temperature extracts 1 hour；
3) leaching liquor is evacuated in liquid storage cylinder by filter, residue is collected back in extraction cylinder, and 1000ml water is added, adds
Heat is slowly stirred to 40-45 DEG C, and low temperature extracts 1 hour；
4) leaching liquor is evacuated in liquid storage cylinder by filter, after mixing, ethyl alcohol will be added for leaching liquor three times
1000ml；
5) leaching liquor that will joined ethyl alcohol is concentrated under reduced pressure into the one third of stoste, obtains skirt by Rotary Evaporators
Band algae concentrate, as component 2.
3. pair algae compounding: component 1 is compounded with the 1:1 in mass ratio of component 2, obtains double algae compound extracts.
A kind of double algae compositions of embodiment 2
The present embodiment provides a kind of double algae compositions, the preparation method is as follows:
By the double algae compound extracts of 1 gained of embodiment by countercurrent spray equipment, it is sufficiently mixed with cyclodextrin, forms ring paste
The double algae compositions of the off-white color that spermatophore is wrapped up in, can be used as anti-aging raw material.Compliance test result
Subjects: the anti-aging essence skincare product as made from 1 gained of embodiment double algae compound extracts, specific preparation side
Method is as follows:
Composition list 1.
Table 1
Technique summary:
(1) A phase raw material is sequentially added in reaction kettle, opens stirring, and begin to warm up, until 85 DEG C, 15min is kept the temperature, is opened
Begin cooling, is down to 60 DEG C, it is spare；
(2) it by after B phase surplus stock premix, is added in A phase, stirring 5min, unlatching homogeneous, 2000 turns, 2min；
(3) 45 DEG C are cooled to hereinafter, sequentially add C phase, is stirred evenly；
(6) 38 DEG C are cooled to bottom discharge.(pH:5.5-7.5 is sieved with 100 mesh sieve).
One, stability is evaluated and tested
(1) instrument and equipment
DGG-9140A electric heating constant-temperature blowing drying box, BCD-196H Rongshida refrigerator.
(2) project is evaluated and tested
A: heat-resisting:
DGG-9140A electric heating constant-temperature blowing drying box is adjusted to 45 DEG C, sample to be tested is put into, is taken out after 24 hours, it is extensive
After multiple room temperature, phenomena such as no discoloration, bleed, layering.
B: cold-resistant:
Sample to be tested is put into Rongshida's freezer chamber of refrigerator (- 10 to -15 DEG C of temperature), is taken out after 24 hours, is restored
After room temperature, no discoloration, bleed, layering phenomena such as.
Two, functional test
1) test index: skin moisture content MMV value；Skin brightness (whiteness) L* value；ITA ° of skin brightness value；Skin
Melanin content MI value；Skin elasticity R2, Q1 value；Image comparison；Image analysis (crow's foot analysis)；
Test crowd: the age 35~55 years old, gender was random；
Test zone: cheek (eye middle line and wing of nose intersection)；Canthus；Face；
Test environment: 22 DEG C ± 1 DEG C of temperature；Relative humidity 50%RH ± 10%RH；
It pays a return visit the time: the 0th week, use the 2nd week after product and the 4th week；
Result formats: data analysis；Image comparison；
2) skin moisture content tester Corneometer CM 825 (German CK company)；
Skin shine probe and Multi probe skin measurement system GL 200 and MPA 9 (German CK company)；
Skin chromatism test probe and Multi probe skin measurement system CL 400 and MPA 10 (German CK company)；
Dermal melanin and ferroheme tester Mexameter MX 18 (German CK company)；
Skin elasticity tester MPA 580 (German CK company)；
Face-image analysis system VISIA-CR (Canfield company, the U.S.)；Image procossing enhances analysis systemPlus Chinese Version 7.0.1 (MediaCybernetics company, the U.S.).
3) test result
A. skin moisture content MMV value
Skin moisture content MMV value, value is bigger, and moisture content is bigger；Conversely, moisture content is smaller.
Hydration rate, i.e. change rate, this value is higher, and moisturizing effect is more obvious.
As shown in Figure 1, within 4 weeks test periods, subject use test sample after, test area skin moisture content with
Sample is gradually risen using the time, be above at the 2nd week and the 4th week before use, and there are significant difference (p < 0.05), it is (each
Time point compares, and " p < 0.05 * " indicates and there were significant differences in the 0th week)；
Skin hydration rate is positive value at the 2nd week and the 4th week, and is increased with sample using the time and increased；
Described above, test sample can significantly improve skin moisture content MMV value, have the effect for improving moisture content of skin
Fruit, and using showing positive effect (p < 0.05) after sample 2 weeks, lasts promote (p < 0.05) after continuous use 4 weeks.
B. ITA ° of skin brightness value
ITA ° of value is the numerical value of the relevant characterization skin brightness of L* and b*.ITA ° of value is bigger, and skin is brighter, conversely,
Skin is more obscure.
ITA ° of value change rate, this value is higher, and skin more tends to be bright.
As shown in Figure 2, within 4 weeks test periods, after subject uses test sample, TA ° of test zone skin I value is with sample
Product are gradually risen using the time, are above at the 2nd week and the 4th week before use, and there are significant difference (p < 0.05)；(when each
Between point compare, " p < 0.05 * " indicate with there were significant differences within the 0th week)；
ITA ° of value change rate is positive value at the 2nd week and the 4th week, and is increased with sample using the time and increased；
Described above, test sample has the effect of improving TA ° of skin I, and using showing positive effect after sample 2 weeks
(p < 0.05), lasts promote (p < 0.05) after continuous use 4 weeks.
C. dermal melanin content MI value
MI value characterizes melanin content in skin, and measurement numerical value is higher, illustrates that the content of melanin is higher.
MI value change rate, this value is lower, and the effect that sample reduces melanin content in skin is better.
From the figure 3, it may be seen that within 4 weeks test periods, after subject uses test sample, test zone dermal melanin content
MI value is gradually decreased with sample using the time, before the 2nd week and the 4th week are below use；
MI value change rate is negative value at the 2nd week and the 4th week, and is increased with sample using the time and reduced；
Described above, test sample has the effect of desalinating dermal melanin.
D. skin whiteness (brightness) L* value
It is white balance that L value, which characterizes L*, and value is bigger, and color is more biased to white, conversely, being biased to black.
L* value change rate, this value is higher, and skin more tends to " brilliant white ".
As shown in Figure 4, within 4 weeks test periods, after subject uses test sample, test zone skin whiteness is (bright
Degree), (each time point compares, and " p < 0.05 * " indicates and there were significant differences in the 0th week)；
L* value is gradually risen with sample using the time, is above at the 2nd week and the 4th week before use, and that there are conspicuousnesses is poor
Different (p < 0.05)；
Skin whiteness (brightness) L* value change rate is positive value at the 2nd week and the 4th week, and is increased with sample using the time
And it increases；
Described above, test sample has the effect of improving skin whiteness (brightness) L* value, and using i.e. aobvious after sample 2 weeks
Existing positive effect (p < 0.05), lasts promote (p < 0.05) after continuous use 4 weeks.
E. skin elasticity R2 value
For skin elasticity R2 value closer to 1, skin elasticity is better.
R2 value change rate is higher, and the effect for improving skin elasticity is better.
As shown in Figure 5, within 4 weeks test periods, subject use test sample after, test zone skin elasticity R2 value with
Sample is gradually risen using the time, be above at the 2nd week and the 4th week before use, and when at the 4th week there are significant difference (p <
0.05)；(each time point compares, and " p < 0.05 * " indicates and there were significant differences in the 0th week).
Skin elasticity R2 value change rate is positive value at the 2nd week and the 4th week, and is increased with sample using the time and risen
It is high；
Described above, the effect that test sample increases skin elasticity is obvious (p < 0.05).
F. skin elasticity Q1 value
For skin elasticity Q1 value closer to 1, skin elasticity is better.
Q1 value change rate is higher, and the effect for improving skin elasticity is better.
By Fig. 6, Fig. 7 it is found that within 4 weeks test periods, after subject uses test sample, test zone skin elasticity Q1
Value is gradually risen with sample using the time, is above at the 2nd week and the 4th week before use, and there are significant differences when at the 2nd week
(p<0.05)；(each time point compares, and " p < 0.05 * " indicates and there were significant differences in the 0th week).
Skin elasticity Q1 value change rate is positive value at the 2nd week and the 4th week, and is increased with sample using the time and risen
It is high；
Described above, the effect that test sample increases skin elasticity is obvious (p < 0.05).
G. Whitening, spot effect
(number 03) test zone of subject 1 gained image under natural light 2 and UV light source is as shown in Figure 8.
As seen from Figure 8, within 4 weeks test periods, after subject 1 (number 03) uses test sample, VISIA-CR
Subject's skin of face whiteness and the colour of skin uniformity gradually rise in 2 image of natural light, are better than using before sample, illustrating to test
Sample has the effect of brilliant white skin, even skin tone；
It was gradually become shallower as at the 0th week by the potential blackspot of skin that red circle is marked in UV Spot image, and is used with sample
Time gradually desalinates, and illustrates that test sample has the effect of desalinating the potential blackspot of skin.
(number 04) test zone of subject 2 gained image under natural light and UV light source: it as seen from Figure 9, is surveying
It tries in 4 weeks periods, after subject 2 (number 04) uses test sample, subject's skin of face in 2 image of VISIA-CR natural light
Whiteness and the colour of skin uniformity gradually rise, and is better than using before sample, illustrating that test sample has brilliant white skin, even skin tone
Effect；
It was gradually become shallower as at the 0th week by the potential blackspot of skin that red circle is marked in UV Spot image, and is used with sample
Time gradually desalinates, and illustrates that test sample has the effect of desalinating the potential blackspot of skin.
H. desalinate microgroove effect
(number 05) test zone of subject 3 gained image under 2 light source of natural light, is shown in Figure 10.
As seen from Figure 10, within 4 weeks test periods, after subject 3 (number 05) uses test sample, VISIA-CR
Region eye circumference microgroove gradually becomes shallower as in 2 image of natural light, and dermatoglyph is gradually smooth-out, the skin lines being labeled in region
It is shortened as sample is presented using the time by length, trend from depth to shallow, illustrates that test sample has the effect for improving desalination microgroove
Fruit.
(number 06) test zone of subject 4 gained image under natural radiant, is shown in Figure 11.
As seen from Figure 11, within 4 weeks test periods, after subject 4 (number 06) uses test sample, VISIA-CR
Periocular area microgroove gradually becomes shallower as in 2 image of natural light, and dermatoglyph is gradually smooth-out, the skin lines being labeled in region
It is shortened as sample is presented using the time by length, trend from depth to shallow, illustrates that test sample has the effect for improving desalination microgroove
Fruit.
I.VISIA-CR image analysis (crow's foot analysis)
Image Acquisition is carried out to subject using facial image analysis system VISIA-CR, and with image procossing enhancing point
Analysis systemPlus Chinese Version 7.0.1 analyzes facial crow's foot.
Wrinkle area accounting is the percentage of the area occupied area domain gross area of wrinkle in selected areas, and wrinkle area accounting is got over
It is low, illustrate that the effect of sample desalination microgroove is better.
The change rate of wrinkle area accounting is lower, and the effect that sample desalinates microgroove is better.
The variation tendency and change rate of subject canthus test zone wrinkle area accounting, are shown in Figure 12,13.By Figure 12, figure
13 it can be concluded that, within 4 weeks test periods, subject use test sample after, canthus test zone wrinkle area accounting is with sample
Product are gradually decreased using the time, before the 2nd week and the 4th week are below use；
Wrinkle area accounting change rate is negative value at the 2nd week and the 4th week, and is increased with sample using the time and dropped
It is low.
Described above, test sample can reduce crow's foot area accounting, have the effect of desalinating microgroove.
Three, double algae compound extracts recombinate effect evaluation in epidermis model in people, are analyzed by quantitative fluorescent PCR
Klotho gene expression and the expression of P63.
1- cultivates principle
People source fibroblast kind (is changed in 0.5 square centimeter of polycarbonate filter, and using supplementing culture medium
Good MCDB153) culture.Cell is cultivated 14 days in gas/liquid interface, is replaced 1 time within culture medium every two days.After culture 14 days,
Epidermis is formed, and can be used for subsequent experimental.
2- Study of cytotoxicity
Cytotoxicity experiment is time of contact in order to determine sample treatment and will not cause cell to epidermis using concentration
Toxicity.
2.1- recombinates epidermis grouping
Product contacts epidermis 24 hours, and every group of sample has 3 repetitions.
Group 1: control (untreated)
Group 2: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 1%)
Group 3: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 2%)
Group 4: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 3%)
2.2- evaluates cell activity by histological stain
Paraffin section is dyed using Goldner variant Masson three-color process, observes cell viability
Influence of 2.3-New Era (Q/S) sample to p63 and klotho gene expression
2.3.1- immunohistochemical analysis P63, immune labeled and picture quantitative analysis
Product contact recombination epidermis 24 hours, every group of sample had 3 repetitions.
2.3.1.1- sample is grouped
Group 1: control (untreated)
Group 2: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 1%)
Group 3: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 2%)
Group 4: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 3%)
2.3.1.2-P63 immunohistochemistry
Use the p63 albumen (Dako, ref:M7317, cloned DAK-p63) of p63 antibody label paraffin section.With
PBS-BSA (0.3%)-Tween20 (0.05%) 1:200 dilution, is incubated at room temperature 1 hour, uses Vectastain RTU
Universal VECTOR streptomysin/peroxidase systematic dyeings simultaneously use VIP colour developing (Vector, SK-4600).Immune dye
Color uses active immunity dyeing instrument (Dako, Autostainer Plus).Micro- sem observation and image quantitative analysis are studied.
2.3.2-RT-PCR the expression of klotho gene is analyzed
Product contact recombination epidermis 24 hours, every group of sample had 3 repetitions.
2.3.2.1- sample is grouped
Group 1: control (untreated)
Group 2: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 1%)
Group 3: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 2%)
Group 4: sample treatment Q/S (the double algae compound extracts of 1 gained of embodiment containing mass percent 3%)
2.3.2.2- using the expression of PCR analysis klotho gene
After sample treatment epidermis 24 hours, the expression of klotho is analyzed:
Cell total rna is extracted using Qiagen kit.Qualitative and quantitative analysis RNA is to ensure its integrality.Use 1% agar
RNA is separated under the conditions of sugared gel 80V, to distinguish 28s and 16s rRNA, tRNA and m RNA (qualitative test).1 μ of reverse transcription
g RNA.Using SuperArray kit, it is cDNA by RNA reverse transcription and further expands.Use fluorescence probe
(Sybergreen), the combination of real-time monitoring detection base.Result is analyzed using corresponding software.
3- experimental result
3.1- epidermal shape studies the cytotoxicity of product to be measured
Experiment is repeated 3 times to ensure epidermis vigor.
Conclusion: as shown in Figure 14, using by be 1%, 2% and 3% containing mass percentage concentration the double algaes of the gained of embodiment 1
The anti-aging essence skincare product of compound extract handles epidermis, and cell viability is good, with the epidermis control group that does not handle without obvious
Difference.
3.2- immunohistochemical analysis P63, immune labeled and picture quantitative analysis
OlympusD software analyzes each group of other picture.
It the results are shown in Table 2, Figure 15.
Table 2
Conclusion: table 2, Figure 15 use as the result is shown by be 1% containing mass percentage concentration, 2% and 3% 1 institute of embodiment
The anti-aging essence skincare product for obtaining double algae compound extracts handles epidermis, and the expression of p63 increases.
The expression of 3.3-RT-PCR transcriptional level analysis Klotho gene
All groups of other Klotho gene expression doses are analyzed using RT-PCR transcriptional level.It the results are shown in Table 3 and Figure 16.
Table 3
Conclusion: table 3 and Figure 16 keratinocyte not table after test sample (1%, 2% and 3%) processing as the result is shown
Up to FGF23.However, it has been found that klotho gene obviously raises, 14%, 20% and 31% is increased separately.Klotho gene tool
Play the role of indicating anti-aging, and is the receptor of FGF23.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of double algae compound extracts, which is characterized in that by Chlamydomanas nivalis extract and opotism algae extract with mass ratio 1:
(1-10) composition.
2. double algae compound extracts according to claim 1, which is characterized in that the Chlamydomanas nivalis is selected from Switzerland's high mountain
Extremophile microalgae.
3. double algae compound extracts according to claim 1 or 2, which is characterized in that the opotism algae is selected from the big of France
Seaweed.
4. double algae compound extracts according to claim 1 to 3, which is characterized in that the Chlamydomanas nivalis extract
Preparation includes: that the acquisition of Chlamydomanas nivalis, culture, homogenate, water are mentioned, separated；
Wherein, the acquisition selection acquires Chlamydomanas nivalis sample in -10 DEG C or less in winter and 10 DEG C of spring or more of period.
5. double algae compound extracts according to claim 4, which is characterized in that the environment temperature of the culture be -30 DEG C -
30 DEG C of adjustable extents, ambient humidity are 10-90%RH adjustable extent；
The environment of the culture must also have illumination linear regulating function, and LED cold light source is red 620nm, indigo plant 460nm.
6. -5 any double algae compound extracts according to claim 1, which is characterized in that the system of the opotism algae extract
Standby includes: selection opotism algae root, is crushed, water is mentioned, filtered；Filter residue adds flooding, repetition secondary, merges filtrate three times；It is added
Ethyl alcohol, rotary evaporation concentrate the filtrate to 2/3rds of volume.
7. -6 any double algae compound extracts according to claim 1, which is characterized in that in the Chlamydomanas nivalis extract
In the preparation process of the opotism algae extract, Extracting temperature is at 60 DEG C or less.
8. application of any double algae compound extracts of claim 1-7 in anti-aging product.
9. a kind of double algae compositions, which is characterized in that contain any double algae compound extracts of claim 1-7.
10. double algae compositions according to claim 9, which is characterized in that double algae compositions further include auxiliary material, preferably
One of cyclodextrin, silica, diatomite are a variety of.
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