CN109925501A - It is a kind of for improving the composition of skin barrier - Google Patents
It is a kind of for improving the composition of skin barrier Download PDFInfo
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- CN109925501A CN109925501A CN201910342140.XA CN201910342140A CN109925501A CN 109925501 A CN109925501 A CN 109925501A CN 201910342140 A CN201910342140 A CN 201910342140A CN 109925501 A CN109925501 A CN 109925501A
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Abstract
The present invention provides a kind of for improving the composition of skin barrier, and the composition includes docosahexaenoic acid and protein.
Description
Technical field
The poly- egg of silk can be improved in the present invention relates to a kind of composition comprising docosahexaenoic acid and protein, the composition
White expression, so as to improve skin barrier.
Background technique
Atopic dermatitis is a kind of chronic skin inflammation by caused by epithelium skin barrier and cutin disdifferentiation.Silk is poly-
Important albumen of the albumen as skin barrier, if will lead to the generation of skin disease, especially if it expresses deficiency in skin
It is atopic dermatitis.
Keratinocyte is the main cell of epithelium, and it is anti-that cuticula, which is the final stage of Keratinocyte differentiation,
The main barrier that only external substance enters human body and internal water is lost.Can be expressed in its atomization a polyprotein (FLG),
Loricrin (LOR), involucrin (IVL) etc., the connection that intersects of these albumen form hornification coating, are maintaining cell skin
It is played an important role in epithelium screen barrier.
Silk polymeric protein is also known as microfilament cohesion albumen, is a kind of alkaline protein for being widely present in skin epidermis.It is main
Wanting function is interacted with keratin intermediate filament, and the aggregation of keratin is cooperateed with, to play the function of the barrier of maintenance skin.
In recent years, deepening continuously with the research to silk polymeric protein gene (FLG), more and more evidences are shown, the gene
Mutation it is related to the generation of a variety of skin diseases.The relationship of FLG gene disease associated therewith is specified, can not only be deepened to this
The understanding of disease, but also the expression of FLG gene can be changed from gene level to improve the barrier function of skin.
The one kind of fatty acid as PPAR agonist can stimulate the metabolism of cytolipin, and silk polyprotein is as a kind of energy
The integrality of skin barrier, skin pH are adjusted while being activated by PPAR agonist and express upregulated protein, UV resists energy
Power and atopic dermatitis morbidity play very important effect.According to the literature, silk polyprotein (FLG) protein abnormal expression causes
The destruction of skin barrier function may be to cause one of infant skin disease pathogenesis, and fatty acid can promote from gene level
The expression of barrier albumen, so as to play an important role in improving abnormal skin barrier function.Martina S's
Researches show that the loss of FLG is enough to change the table of albumen relevant to atopic dermatitis pathogenesis in the case where no inflammation
Up to level.These include adjusting inflammation, the protein of proteolysis and cytoskeleton function.
The prior art, which reports docosahexaenoic acid, can promote FLG expression.But docosahexaenoic acid is as activity
Ingredient is proved that the tolerance in cell toxicity test is poor when being used alone.
Summary of the invention
Present inventor has been surprisingly found that protein can be synergistic with docosahexaenoic acid, to improve cell
The expression of silk polyprotein.Therefore, inventor develops a kind of composition comprising docosahexaenoic acid and protein, and confirms
The composition has better tolerance relative to exclusive use docosahexaenoic acid in cell toxicity test.Meanwhile institute
The composition comprising docosahexaenoic acid and protein is stated compared with docosahexaenoic acid is used alone as active constituent
Compared with, it can be while largely promoting the expression of cytofilament polyprotein, improving safety, 22 carbon of further promotion
Efficiency of the acid in the expression of cytofilament polyprotein, so as to improve skin barrier.
Sea-mussel mucin is a kind of secretion byssus of marine mussel, also referred to as mussel byssus protein, is that seashells purple is made a gift of
The special protein of one kind of the secretions such as shellfish, Trachyostracous mussel, Perna viridis, has high-intensitive, high tenacity and high waterproofness.Although
Sea-mussel mucin has the above performance, but its product applications is very limited, at present mainly with sea-mussel mucin solution or jelly
Dry powder formulations are applied to microcosmic cell adherence and tissue adhesive.
Sea-mussel mucin used in the present invention is its form of extract, is directly purchased from Bei Ruisen company.
Therefore, on the one hand, the present invention provides a kind of for improving the composition of skin barrier, and the composition includes 20
Two carbon acids and protein are as active constituent.
According to a preferred embodiment, the content of the docosahexaenoic acid is 0.0001% to 10% (W/V),
It is preferred that 0.0001% to 5% (W/V), more preferable 0.0001% to 1% (W/V), even more preferably 0.0001% to 0.1% (W/
V), most preferably 0.005% to 0.05% (W/V), and wherein the content of the protein is 0.1%-20% (W/V).
According to a preferred embodiment, the content of the docosahexaenoic acid is 10 μM -100 μM.
According to a preferred embodiment, the protein is selected from sea-mussel mucin, sea-mussel mucin extract or white
Albumen.The sea-mussel mucin extract includes sea-mussel mucin hydrolyzed peptide and other hydrolysates.
According to most preferred embodiment, the composition that the present invention is used to improve skin barrier includes docosahexaenoic acid
With sea-mussel mucin hydrolyzed peptide as active constituent.
According to a preferred embodiment, the docosahexaenoic acid is saponified.Saponification of the present invention
Refer to docosahexaenoic acid and alkali reaction, generates the reaction of docosahexaenoic acid salt.The saponification is by by 22 carbon
Acid and saponification agent carry out.Conventional saponification agent is highly basic, including KOH and NaOH.Preferred saponification agent is NaOH.When 20
After two carbon acids and sodium hydroxide carry out saponification, docosahexaenoic acid correspondingly generates the sodium of docosahexaenoic acid
Salt.
In the present invention, docosahexaenoic acid and protein are used as active constituent for improving cytofilament polyprotein
Expression, so as to improve skin barrier.The present composition can need to be prepared into different dosage forms, preferably external preparation for skin system according to different
Agent, including liquid agent, gelling agent, emulsion, creme, foaming agent, patch etc..However, it will be appreciated by those skilled in the art that institute of the present invention
State composition can according to the various dosage forms of composition add it is various needed for additives, including but not limited to moisturizer, emulsifier,
Antioxidant and pH adjusting agent etc..In the present invention, the moisturizer can be selected from glycerol, hydrolysis Sodium Hyaluronate or hyalomitome
Sour sodium, polyethylene glycol etc.；The emulsifier can be selected from Cremophor RH40；The antioxidant can be selected from fertility
Phenol, glutathione etc.；The pH adjusting agent can be selected from sodium citrate or citric acid.The present composition can also be according to combination
The various dosage forms of object add various required excipient.For example, when the present composition is prepared into liquid agent, the excipient
It can be selected from water, physiological saline etc.；When the present composition is prepared into gelling agent, the excipient can spread out selected from cellulose
Biology, starch, gelatin, agar, polysaccharide etc.；When the present composition is prepared into creme, the excipient can selected from glycerol,
Vaseline, paraffin etc.；When the present composition is prepared into foaming agent, the excipient can be selected from hydroxypropyl methyl fiber
Element, polyethylene glycol lauryl sodium sulfate, polyoxyethylene fatty alkyl ether sulfonate etc.；When the present composition is prepared into patch
When, the excipient can be selected from cellulose derivative, starch, gelatin, agar, polysaccharide etc..
The composition for being used to improve skin barrier of the invention and various dosage forms can be according to known to those skilled in the art
Conventional method prepared.
On the other hand, the present invention is provided to improve the composition of skin barrier in preparation for treating atopic dermatitis
Purposes in drug.
Unexpectedly, inventors be surprised to learn that protein especially sea-mussel mucin extract can be with 22 carbon six
Olefin(e) acid is synergistic, to improve the expression of cytofilament polyprotein, improves skin barrier, to treat atopic dermatitis.
The advantages of present invention is for improving the composition of skin barrier is:
1. there is preferable tolerance relative to exclusive use docosahexaenoic acid in cell toxicity test；
2. be used alone docosahexaenoic acid as active constituent compared with, the composition can be largely
Ground promotes the expression of cytofilament polyprotein and improves safety；
3. the protein and docosahexaenoic acid are synergistic, docosahexaenoic acid is further promoted in cytofilament
Efficiency in polyprotein expression.
Detailed description of the invention
Fig. 1 shows blank control, docosahexaenoic acid, mixture, " docosahexaenoic acid-sea-mussel mucin is extracted
Object ", docosahexaenoic acid-Na and mixture " docosahexaenoic acid-Na- sea-mussel mucin extract " are to cell monolayer
The influence of FLG gene by fluorescence quantitative PCR result.
Fig. 2 indicates that " docosahexaenoic acid-sea-mussel mucin extracts for blank control, docosahexaenoic acid, mixture
Object ", docosahexaenoic acid-Na and mixture " docosahexaenoic acid-Na- sea-mussel mucin extract " are to 2D cellular immunity
The influence of fluorescence results.
Fig. 3 indicates that " docosahexaenoic acid-sea-mussel mucin extracts for blank control, docosahexaenoic acid, mixture
The 2D cell fluorescence of object ", docosahexaenoic acid-Na and mixture " docosahexaenoic acid-Na- sea-mussel mucin extract "
Expression intensity.
Fig. 4 indicates the 3D cell smearing knot of reference composition A, prior art compositions B and C, the present composition D and E
Fruit.
Fig. 5 indicates the 3D skin model of reference composition A, prior art compositions B and C, the present composition D and E
Luciferase expression intensity.
Fig. 6 indicates docosahexaenoic acid, the mixture " docosahexaenoic acid-sea-mussel mucin of addition various concentration
The cell of extract ", docosahexaenoic acid-Na and mixture " docosahexaenoic acid-Na- sea-mussel mucin extract " is living
Force value.
Specific embodiment
Embodiment 1: docosahexaenoic acid+serum-free bovine albumin
20% serum-free bovine albumin of 20g is prepared, in 100ml phosphate buffer with 2000xg, 4 DEG C of centrifugations, 15 points
After completely dissolution, 10 μM of docosahexaenoic acids are added in clock, and 65 DEG C of water-baths are mixed 30 minutes, are distributed into after 0.22 μm of film filtering
1.5ml sterile centrifugation tube is spare.
Embodiment 2: docosahexaenoic acid+sea-mussel mucin extract
20% sea-mussel mucin extract of 20g is prepared, in 100ml phosphate buffer with 2000xg, 4 DEG C of centrifugations, 15
After completely dissolution, 10 μM of docosahexaenoic acid is added in minute, and 65 DEG C of water-baths are mixed 30 minutes, dispensed after 0.22 μm of film filtering
Enter 1.5ml sterile centrifugation tube, this mixture is known as " docosahexaenoic acid-sea-mussel mucin extract ".
Embodiment 3: the preparation of docosahexaenoic acid-Na
The DMSO solution 1mL for taking docosahexaenoic acid to be configured to 200 μM, while the NaOH solution 1mL of 200uM is configured,
Two solution are mixed with 1:1 ratio, are mixed under the conditions of 65 DEG C, 500RPM, until be cooled to room temperature after transparent clarification,
It is distributed into 1.5mL sterile centrifugation tube after 0.22 μM of film filtering, i.e. acquisition docosahexaenoic acid-Na.
Embodiment 4: docosahexaenoic acid+NaOH+ sea-mussel mucin extract
20% sea-mussel mucin extract of 20g is prepared, in 100ml phosphate buffer with 2000xg, 4 DEG C of centrifugations, 15
After completely dissolution, 10 μM of docosahexaenoic acid and the NaOH mixed aqueous solution of equimolar volume, 65 DEG C of water-baths are added in minute
It mixes to solution and clarifies, be distributed into 1.5ml sterile centrifugation tube after 0.22 μm of film filtering, this mixture is known as " two dodecahexaenes
Acid-Na- sea-mussel mucin extract ".
Embodiment 5: the composition (data unit is % (W/V) in table) according to shown in the following table 1 prepares reference composition
A, prior art compositions B and C and the present composition D and E.
Table 1
The preparation method of composition A-E is specific as follows:
Add water in modulation kettle, is heated to 80 DEG C of heat preservations.By 99.5% glycerol and Sodium Hyaluronate in dry stainless steel
It is pre-dispersed in bucket, then put into modulation kettle, it dissolves it sufficiently within 30 minutes with 30rmp stirring, during which keeps the temperature at 75-80
℃.By docosahexaenoic acid or docosahexaenoic acid-Na salt and Cremophor RH40 and tocopherol it is dry not
Predissolve in rust steel drum, then put into and be cooled in 40 DEG C of modulation kettles below, it is made it dissolve within 10 minutes uniformly with 30rmp stirring.
Then modulation kettle is added in sea-mussel mucin extract, stirring makes it dissolve uniformly for 10 minutes.Then it samples, surveys pH at 25 DEG C
=5.5 ± 0.50, sodium citrate and citric acid is added, discharges after 0.2 μm of film filtering filling.
Embodiment 6: cell monolayer gene expression test
By blank control, docosahexaenoic acid, mixture " docosahexaenoic acid-sea-mussel mucin extract ", two
Dodecahexaene acid-Na and mixture " docosahexaenoic acid-Na- sea-mussel mucin extract " (contain with Eplife culture medium
HKGS and 1.5mM calcium chloride) mixing after cultivate 24 hours after, the extraction of total serum IgE is carried out with Trizol, with RT-qPCR detection FLG
Changes in gene expression.Specific method: cell each adds the Trizol reagent of 1ml in 6 orifice plates, blows and beats, is transferred to without miscellaneous repeatedly
In the ep pipe of the 1.5ml of nucleic acid and nuclease, the chloroform of 200 μ l is added, vortex 1min stands 1-2min, 12000g, 4 DEG C, from
Heart 10min draws supernatant into new centrifuge tube, isometric isopropanol is added.It is vortexed, 12000g, 4 DEG C, 10min, carefully
Take out supernatant, be added the 75%DEPC ethyl alcohol of 1ml, 9000g, is centrifuged 5min by 4 DEG C, and careful takes out ethyl alcohol, is added 50 μ l's
Without enzyme water, after carrying out RNA quantitative adjusting concentration with Qubit 3.0, it is using Reverse Transcription and poly (A) primed reverse transcription
cDNA.According to actual requirement, cDNA concentration is adjusted to 25ng μ l-1, primer needed for taking 50ng to mix, and carry out fluorescent quantitation
PCR.Correction loading errors are carried out using GAPDH as reference gene, (specific method refers to the expression quantity of calculating FLG mRNA
The FastStart Essential DNA Green Master of Roche, the 6th edition, can be under the official website of Roche Diagnistics
It carries and obtains).
Table 2. is used for the cDNA design of primers of real-time fluorescence quantitative PCR
The results are shown in attached figure 1.In the experiment of the quantitative fluorescent PCR of cell monolayer, compared with blank control group, 25 μM 20
FLG gene expression can be promoted 1.78 times by the application of two carbon acids, and work as docosahexaenoic acid and sea-mussel mucin
After mixing application, promotes effect and be further enhanced, there are about 2.66 times of promotions.
After docosahexaenoic acid and sodium hydroxide carry out saponification, FLG is able to ascend under 100 μM of administration dosage
2.45 times of expression, after the sodium salt of docosahexaenoic acid and sea-mussel mucin combine after applying saponification, expression is promoted also into one
Step increases to 2.7 times (experiment primer sequence is as described in Table 2, carries out homogenization processing by internal reference of GAPDH).To sum up result institute
It states, is able to ascend docosahexaenoic acid in the gene expression efficiency increase 25%- of FLG with being used in combination for sea-mussel mucin
88%.
Embodiment 7:2D cellular immunofluorescence result
It grows cell on glass cover-slip, at room temperature, is incubated in 4% paraformaldehyde (being dissolved in PBS, pH 7.4)
Cell 10 minutes.It is washed cell 3 times with ice PBS.With PBS (X-100 containing 0.25%Triton) samples of incubation 10 minutes.
Triton X-100 is for improving the most common detergent of antibody permeability.It is washed cell 3 times, every time 5 minutes with PBS.With
PBST (PBS+0.1%Tween20) of 1%BSA, 22.52mg/mL glycine incubated cell 30 minutes, the non-spy of blocking antibody
The opposite sex combines.At room temperature, it is incubated in wet box with dilution antibody rabbit-anti silk polyprotein 1:500 dilution (being dissolved in the PBST of 1%BSA)
Hatching cell 1 hour.Solution is poured out, is washed cell 3 times, every time 5 minutes with PBS.At room temperature, with secondary antibody goat antirabbit igG 1:
5000 dilutions (being dissolved in 1%BSA) are protected from light incubated cell 1 hour.Two corresponding anti-solution is poured out, is protected from light washing cell 3 times with PBS, often
Secondary 5 minutes.Green fluorescence intensity is observed under the microscope.And it is analyzed using imagej.
Attached drawing 2 shows 2D cellular immunofluorescence result.The result shows that: compared with blank control group, two dodecahexaenes
The application of acid, which can express FLG protein fluorescence, improves 2.58%, and when docosahexaenoic acid and sea-mussel mucin are mixed
After closing application, promotes effect and be further enhanced, there are about 30.44% promotions.
After docosahexaenoic acid and sodium hydroxide carry out saponification, the expression of FLG protein fluorescence promotes 22.65%,
After the sodium salt of docosahexaenoic acid and sea-mussel mucin combine after applying saponification, expression promotes 47.26%.To sum up result institute
It states, docosahexaenoic acid can be made to increase in the protein fluorescence expression efficiency of FLG with being used in combination for sea-mussel mucin
24.61%-27.86% (fluorescence experiments carry out homogenization processing by internal reference of DAPI dyeing).
Embodiment 8:3D histogenic immunity fluorescence
Experimental group and treatment conditions are grouped and mark, every group of 12 models.Sample sets use the side of surface administration
Formula is handled, and administered volume is 25 μ L/ models.Positive controls are taken and are administered under liquid, and it is female will to shift to an earlier date prepared Wy14643
Culture solution is added in liquid, mixes, and ultimate density is 50 μM.All 6 orifice plates handled well are placed in 37 DEG C, 5%CO2, relative humidity
In 95% incubator, it is incubated for for 24 hours.After being incubated for for 24 hours, 6 orifice plates are taken out from incubator, and different experiments are cleaned using sterile PBS
Group skin model.Each model scavenging period control is in 1min or so, to guarantee the time phase of each model contact PBS
Together.After cleaning 15 times, free surface moisture in model is gently sucked with aseptic cotton carrier, it is rear to place in 6 orifice plates.
Immunofluorescence test:
(1) dewax aquation: paraffin section is placed in dimethylbenzene and impregnates 15min, impregnates 15min again after replacing dimethylbenzene,
5min is impregnated in dehydrated alcohol, and 5min is impregnated in 95% ethyl alcohol, impregnates 5min in 75% ethyl alcohol.PBS buffer solution cleaning 3 times, often
Secondary 5min；
(2) antigen retrieval: being put into 0.01M sodium citrate antigen retrieval solution for paraffin section, cooling using Pressure method
Slice is taken out afterwards.PBS buffer solution cleans 3 times, 5min/ times；
(3) serum is closed: being added dropwise and the homologous serum block of secondary antibody, 37 DEG C of closing 60min, without rinsing；
(4) primary antibody is incubated for: being added dropwise primary antibody working solution (being prepared with confining liquid), 4 DEG C of overnight incubations.PBS buffer solution cleaning 3
It is secondary, 5min/ times.
(5) secondary antibody is incubated for: secondary antibody working solution, 37 DEG C of incubation 1h are added dropwise.PBS buffer solution is cleaned 3 times, 5min/ times.
(6) lining dye: the nucleus after 100 μ L dilution is added dropwise serves as a contrast dye liquor Hoechst 33342, is incubated at room temperature 5min.PBS is slow
Fliud flushing is cleaned 3 times, 5min/ times.
(7) an anti-fluorescence quenching of drop, mounting mounting: is added dropwise.
(8) it takes pictures: room temperature preservation, it is interior for 24 hours to take pictures and handle using fluorescence microscope.
Fig. 4 indicates the 3D cell smearing knot of reference composition A, prior art compositions B and C, the present composition D and E
Fruit and Fig. 5 indicate reference composition A, prior art compositions B and C, the present composition D and E 3D skin model it is glimmering
Light expression intensity.
The result shows that: composition B, C, D and E after addition docosahexaenoic acid are expressed than reference composition A in FLG
On promoted, high dose docosahexaenoic acid composition C has brighter compared with low dosage docosahexaenoic acid composition B
Aobvious FLG facilitation, after adding sea-mussel mucin extract (composition D and E), facilitation reinforces that (upper layer is again
FLG, lower layer are nuclear targeting).
Therefore, docosahexaenoic acid can promote the FLG expression contents in Human keratinocytes, docosahexaenoic acid
Or the combination of docosahexaenoic acid-Na and sea-mussel mucin extract can enhance Docosahexaenoic acid and promote cell FLG
The expression of mRNA.
In all compositions, expresses and promoted after docosahexaenoic acid-Na is combined with sea-mussel mucin hydrolyzed peptide
Effect is maximum.Meanwhile it can be by the 3D cell membrane of docosahexaenoic acid in the composition with being used in combination for sea-mussel mucin
Type FLG protein fluorescence expression efficiency improves 21.17%.
Embodiment 9: cell toxicity test
It is made into individual cells suspension with culture solution is obtained containing 10% tire calf serum, with 1000-10000, every hole cell inoculation
To 96 orifice plates, every 200 μ l of pore volume.With general condition of culture, docosahexaenoic acid, the mixture of various concentration is added in next day
" docosahexaenoic acid-sea-mussel mucin extract ", docosahexaenoic acid-Na and mixture " docosahexaenoic acid-
Na- sea-mussel mucin extract " is cultivated 24 hours.After culture 24 hours, every hole adds MTT solution, and (5mg/ml is prepared with PBS, pH
=7.4) 20 μ l.Continue to be incubated for 4h, careful inhale abandons culture supernatant in hole.Every hole adds 150 μ l DMSO, vibrates 10min, makes to tie
Brilliant object sufficiently melts.490nm wavelength is selected, each hole absorbance value is measured on enzyme linked immunological monitor, is recorded as a result, with sample
Concentration is abscissa, and light absorption value is that ordinate draws cell growth curve and calculates half inhibiting rate concentration.The result shows that: in phase
With using in the case where identical metering, the mixture cell viability of docosahexaenoic acid-Na salt and sea-mussel mucin is better than
The exclusive use of docosahexaenoic acid-Na, while docosahexaenoic acid-Na is better than docosahexaenoic acid.Simultaneously in 25 μ
M to 100 μM comes into operation under concentration, and the use of sea-mussel mucin can make the cytotoxicity in cell viability experiment be reduced by about 10%.
Claims (13)
1. a kind of for improving the composition of skin barrier, the composition includes docosahexaenoic acid and protein.
2. composition according to claim 1, wherein the content of the docosahexaenoic acid is 0.0001% to 10%
(W/V), and wherein the content of the protein is 0.1% to 20% (W/V).
3. composition according to claim 2, wherein the content of the docosahexaenoic acid is 0.0001% to 5%
(W/V)。
4. composition according to claim 3, wherein the content of the docosahexaenoic acid is 0.0001% to 1%
(W/V)。
5. composition according to claim 4, wherein the content of the docosahexaenoic acid is 0.0001% to 0.1%
(W/V)。
6. composition according to claim 1 or 2, wherein the protein is selected from sea-mussel mucin, sea-mussel mucin mentions
Take object or albumin.
7. composition according to claim 1 or 2, wherein the sea-mussel mucin extract is hydrolyzed comprising sea-mussel mucin
Polypeptide.
8. composition according to claim 1 or 2, wherein the docosahexaenoic acid is saponified.
9. composition according to claim 8, wherein the saponification is by carrying out docosahexaenoic acid and saponification agent.
10. composition according to claim 9, wherein the saponification agent is NaOH.
11. composition according to claim 1 or 2, wherein the composition further include moisturizer, it is emulsifier, anti-oxidant
At least one of agent and pH adjusting agent.
12. composition according to claim 11, wherein the moisturizer is selected from glycerol, hydrolysis Sodium Hyaluronate or transparent
Matter acid sodium；The emulsifier is selected from Cremophor RH40；The antioxidant is selected from tocopherol；The pH adjusting agent is selected from
Sodium citrate or citric acid.
13. -12 described in any item compositions for improving skin barrier are answered in preparation for treating spy according to claim 1
Purposes in the drug of property dermatitis.
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| CN110731911A (en) * | 2019-10-17 | 2020-01-31 | 贝亲母婴用品（上海）有限公司 | composition for increasing water content of stratum corneum |
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