CN103505374B - A kind of anti-skin photoage preparation and application thereof - Google Patents
A kind of anti-skin photoage preparation and application thereof Download PDFInfo
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- CN103505374B CN103505374B CN201210206686.0A CN201210206686A CN103505374B CN 103505374 B CN103505374 B CN 103505374B CN 201210206686 A CN201210206686 A CN 201210206686A CN 103505374 B CN103505374 B CN 103505374B
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Abstract
This application discloses a kind of anti-skin photoage preparation containing Royal Jelly acid (10-HDA), meanwhile, also disclose 10-HDA and preparing the application in skin care item, cosmetics and medicine cosmetic.The anti-photoaging formulation that the application provides, can significantly improve the activity of the postradiation cell of UVA, suppresses UVA induction to produce ROS, suppresses the cell ageing of UVA induction, have the effect of significant anti-light aging.The research display of its mechanism of action, can the 10-HDA contained in the application's anti-photoaging formulation suppress p-JNK and p38? the activation of MAPK signal path, reduce the expression of MMP-1 and MMP-3 of UVA induction, thus the collagen content in increase cell, be a kind of preparation of effective anti-skin photoage.
Description
Technical field
The application relates to anti aging effect formulation art, particularly relates to Royal Jelly acid at anti-skin photoage preparation, as the application in anti-light aging skin care item, cosmetics, medicine cosmetic.
Background technology
Skin aging mainly comprises intrinsic aging and extrinsic aging.Extrinsic agingly refer to that skin is subject to such environmental effects and the damped exponential model caused, because of based on the impact of ultraviolet (Ultraviolet, UV) radiation, therefore be also called photoaging.Long wave ultraviolet (UVA) and ultraviolet B radiation (UVB) all can cause photoaging.Because to skin, UVB burns that to act on comparatively UVA strong 1000 times, UVB is considered to the dominant spectral causing photoaging always for a long time.But nearest research shows that UVA is only the dominant spectral causing skin photoage.
The main clinical manifestation of skin photoage is wrinkle of skin, coarse, dry, lax, telangiectasis, fragility increase and pigmentation.Chronic sun exposure can cutaneous various kinds of cell composition and organizational structure change, and wherein the distinctive change of most is the change of dermal extracellular matrix composition.Dermal extracellular matrix comprises collagen fiber, elastic fibers, aminopolysaccharide and proteoglycan etc., and wherein I-type collagen is its major structural protein.At photoaging skin, I type and type III collagen fiber reduce, disorderly, elastic fiber degeneration, thicker, and assemble in bulk, aminopolysaccharide cracking, causes the generation of cutis laxa, wrinkle.
The mechanism of skin photoage is not yet completely clear, but matrix metalloproteinase (Matrixmetalloproteinases, MMPs) expression is increased or increased activity is the important mechanisms that the matrix components such as collagen fiber are degraded.MMPs is one group of zinc protease family, by 25 member compositions such as MMP-1, MMP-2, MMP-3.That soluble protein or membrane bound protein are divided into collagen enzyme, gelatin enzyme, stromelysin and membranous type MMPs after secreting according to its substrate specificity and their.In 19 kinds of MMPs that normal human skin is expressed, three kinds are only had to make a response to UV irradiation and significantly induced, i.e. MMP-1, MMP-3 and MMP-9.After UV irradiates 24 hours, MMP-1 and MMP-3mRNA level significantly increases, and MMP-9mRNA level only has appropriateness to increase.
The MMPs of UV radiation-induced plays an important role in the degradation process of skin photoage dermal extracellular matrix composition.Keratinocyte and the multiple matrix metalloproteinase proenzyme of fibroblasts to secrete can be induced after ultraviolet radiation skin, and by regulating the activation of proenzyme and the expression of matrix metallo-proteinase inhibitor to make it change into activated MMPs.The dermal extracellular matrix composition that the MMPs activated can degrade nearly all, causes the skin photoage such as cutis laxa, wrinkle formation to change.
The dermal matrix albumen that dissimilar MMP degrades different.Such as, MMP-1 degraded I, II and type III collagen protein, MMP-9 degraded IV, V collagen protein and gelatin, MMP-3 then has substrate specificity widely, it can be degraded polytype collagen protein and substrate molecule, as proteoglycan, laminin，LN and fibronectin.For collagen protein, especially I type and type III collagen protein, first start collagen fiber by MMP-1 and rupture, then upborne MMP-3 and MMP-9 degrades further.Therefore, it is excessive that long-term UV irradiation can cause MMPs to produce, and the extracellular matrix components such as degrade collagen albumen, causes the skin photoage such as cutis laxa, wrinkle formation to change then.
In application on human skin, UV can induce the keratinocyte of epidermis and the fibroblast of corium to express MMPs, and wherein keratinocyte is the Major cellular sources of MMPs.UV radiation-induced MMPs gene expression relates to complicated cell-signaling pathways, and wherein most important is exactly MAPK and NF-κ B signal path, and the former is relevant with the activation of transcription factor AP-1, and the latter is relevant with the activation of transcription factor NF-KB.AP-1 and the NF-κ B of activation can be combined with AP-1 and the NF-kB site of multiple MMPs promoter respectively and induce the expression of MMPs.Verified, the AP-1 stimulation keratinocyte of UV activation and transcribing of fibroblast MMP-1 and MMP-3.Because MMP-1 with MMP-3 promoter has similar structure, UVB irradiates postactivated AP-1 and is combined with the AP-1 site of MMP-1 and MMP-3 promoter respectively and causes transcribing of MMP-1 and MMP-3.The generation that UV irradiates the active oxygen (Reactiveoxygenspecies, ROS) caused is considered to the initiating link activating above-mentioned two signal paths.
In sum, UV irradiates the activation by activating complicated signal path induction MMPs, and then degraded dermal extracellular matrix composition causes skin photoage, the initiating link or the key link that therefore block these signal paths just can suppress MMPs to activate the effect reaching anti-light aging.In recent years, MMPs is considered to a promising anti-light aging therapy target.Research and develop effective MMPs inhibitor and become an important study hotspot.Up to the present, the studied exploitation of MMPs inhibitor coming from natural biological in a large number has been had.They respectively with ROS, MAPK path, NF-κ B path or multiple link for target spot reaches the effect of anti-light aging.Be that the MMPs inhibitor of target spot is as white floss Rhizoma Polypodiodis Nipponicae leaf extract, phylloxanthin, little coralline algae etc. with ROS.These materials suppress the expression of MMPs by removing ROS.The MMPs inhibitor being target spot with MAPK path is if fucoidan, trans-zeatin etc. are by blocking the MAPK path of UV mediation to suppress the expression of MMPs.The MMPs inhibitor being target spot with NF-κ B path is as the expression by suppressing the NF-κ B path of UV mediation to suppress MMPs such as cordycepin, magnolol.
Royal Jelly acid (chemical name: 10-HAD, 10-hydroxy-2-decenoicacid, 10-HDA) is a kind of natural materials of the glandular secretion such as lingual gland, mandibular gland of worker bee, is distinctive a kind of unsaturated fatty acid in Lac regis apis.By Germany scientist D.J. youth lattice at nineteen twenty-one Late Cambrian.From then on, the research about Royal Jelly acid is reported successively, discloses Royal Jelly acid and has medical health effect, in acute radiation injury, have significant curative effect.But anti-acute radiation and anti-light aging are diverse concepts, both mechanism of action are also completely different, therefore, not yet have relevant research report at present in the anti-light aging of Royal Jelly acid.
Summary of the invention
The object of the application is to provide a kind of anti-skin photoage preparation newly, and 10-HDA is preparing the application in anti-light aging skin care item, anti-light aging cosmetics, anti-light aging medicine cosmetic.
To achieve these goals, the application have employed following technical scheme:
This application discloses a kind of anti-skin photoage preparation, the main active substances of its anti-light aging comprises Royal Jelly acid.It should be noted that, it will be understood by those skilled in the art that in this anti-skin photoage preparation and also contain other pharmaceutically acceptable carrier, solvent, or also can contain the typical additives etc. of skin protection, skin Caring.
Further, in above-mentioned anti-photoaging formulation, the content of 10-HDA is 0.5-1.5mmol/L.
Disclosed herein as well is the application of above-mentioned anti-skin photoage preparation in anti-light aging skin care item, anti-light aging cosmetics or anti-light aging medicine cosmetic.
Concrete, this application discloses a kind of anti-light aging skin care item, the main active substances of its anti-light aging comprises Royal Jelly acid.
Further, in above-mentioned skin care item, the content of Royal Jelly acid is 0.5-1.5mmol/L.
Concrete, disclosed herein as well is a kind of anti-light aging cosmetics, the main active substances of its anti-light aging comprises Royal Jelly acid.
Further, in above-mentioned cosmetics, the content of Royal Jelly acid is 0.5-1.5mmol/L.
Concrete, disclosed herein as well is a kind of anti-light aging medicine cosmetic, the main active substances of its anti-light aging comprises Royal Jelly acid.
Further, in above-mentioned medicine cosmetic, the content of Royal Jelly acid is 0.5-1.5mmol/L.
Owing to adopting above technical scheme, the beneficial effect of the application is:
This application provides a kind of new anti-photoaging formulation, wherein containing 10-HDA.Experimental result shows, 10-HDA can significantly improve the activity of the postradiation cell of UVA, suppresses UVA induction to produce ROS, suppresses the cell ageing of UVA induction, have the effect of significant anti-light aging.Research display, the 10-HDA contained in the anti-photoaging formulation of the application can suppress the activation of p-JNK and p38MAPK signal path, reducing the expression of MMP-1 and MMP-3 of UVA induction, thus increase the collagen content in cell, is a kind of preparation of effective anti-skin photoage.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present application Royal Jelly acid to the vitality test result through the postradiation cell of UVA;
Fig. 2 is that in the embodiment of the present application, Royal Jelly acid affects result to active oxygen;
Fig. 3 is the anti-light aging effect detection of Royal Jelly acid in the embodiment of the present application;
Fig. 4 is that in the embodiment of the present application, Royal Jelly acid affects result to collagen protein;
Fig. 5 is that the Royal Jelly acid of fluorescence quantitative PCR detection in the embodiment of the present application affects result to the mrna expression of MMP-1;
Fig. 6 is that the Royal Jelly acid of fluorescence quantitative PCR detection in the embodiment of the present application affects result to the mrna expression of MMP-3;
Fig. 7 is that the Royal Jelly acid that in the embodiment of the present application, protein immunoblotting detects affects result to MMP-1 albumen;
Fig. 8 is that the Royal Jelly acid that in the embodiment of the present application, protein immunoblotting detects affects result to MMP-3 albumen;
Fig. 9 is that the Royal Jelly acid that in the embodiment of the present application, protein immunoblotting detects affects result to JNK albumen;
Figure 10 is that the Royal Jelly acid that in the embodiment of the present application, protein immunoblotting detects affects result to p38 albumen.
Detailed description of the invention
The generation that present inventor confirms the cytotoxicity that 10-HDA can suppress UVA to induce and ROS by a series of experiment, the expression suppressing the fibroblast aging of UVA induction, suppress the activation of p-JNK and p38MAPK signal path, reduce MMP-1 and MMP-3 of UVA induction, increase the collagen content in cell conditioned medium liquid.
On the basis of above-mentioned research, present inventor provides a kind of new anti-skin photoage preparation containing 10-HDA.10-HDA adds in medically acceptable solvent carrier (as liquid paraffin etc.) and is prepared from, for the anti-light aging of skin by this anti-skin photoage preparation.
Further, it will be appreciated by those skilled in the art that, in view of the anti-light aging effect of 10-HDA, can easily 10-HDA or above-mentioned anti-skin photoage preparation be added in skin care item, medicine cosmetic or other cosmetics, to prepare containing the skin care item with anti-light aging effect of 10-HDA, medicine cosmetic or other cosmetics.
In addition, 10-HDA, except for except above-mentioned skin care item, cosmetics and medicine cosmetic, it will be understood by those skilled in the art that 10-HDA can also be made into other pharmaceutically acceptable preparations, for treatment or the beauty treatment of anti-skin photoage; Or 10-HDA and other be applicable to the carrier locally applied on skin and be prepared into the compositions with anti-light aging effect.
Also by reference to the accompanying drawings the application is described in further detail below by specific embodiment.Following examples are only further described the application, should not be construed as the restriction to the application.
The anti-light aging effect of embodiment one Royal Jelly acid (10-HDA)
(1) human skin fibroblast is cultivated
Experimental technique: get child and to peritomize postoperative prepuce tissues, is separated the human fibroblasts obtaining purification, by cell culture in containing 10% superfine hyclone (FBS) and mycillin DMEM culture fluid in, be placed in containing 5%CO
237 DEG C of incubators in cultivate.
(2) UVA irradiates
Experimental technique: give UVA when fibroblast is covered with and converges to 80% ~ 90% and irradiate.Cell is taken out from incubator, inhales and abandon culture medium, wash 2 times with the phosphate buffer (PBS) of 37 DEG C of preheatings.Every hole adds the thin layer PBS of equivalent, then irradiates with the UVA of various dose.Inhale immediately after irradiation and abandon PBS solution, add freshly prepared normal culture fluid, be placed in the humidity of 95%, 5%CO
237 DEG C of incubators in cultivate.In this example, normal culture fluid is namely containing the superfine hyclone (FBS) of 10% and the DMEM culture fluid of mycillin.
(3) cell viability measures
Experimental technique: in order to detect the cytotoxicity whether 10-HDA can suppress UVA to induce, this example CCK-8 reagent (Dojindo, Japan) measures cell viability.Experiment is divided into 3 groups: blank group, UVA irradiation group and 10-HDA group, and often group establishes 3 multiple holes.Blank group: normal culture fluid is cultivated; UVA irradiation group: use 4J/cm
2uVA irradiates, and changes normal culture fluid and cultivate after irradiation; 10-HDA group: use 4J/cm
2uVA irradiates, and cultivates after irradiating with the culture fluid containing 1mmol/L10-HDA.By fibroblast by 5 × 10
3cells/well density is inoculated in 96 well culture plates, and 100 μ l culture fluid are added in every hole, and after inoculation 24h, UVA irradiation group and 10-HDA cell give 4J/cm respectively
2uVA irradiates, and after irradiation, each group cell adds containing according to above-mentioned condition or do not contain the culture fluid of 10-HDA, chooses respectively after cultivating 24h, 48h and tests.In every hole, add the CCK-8 reagent of 10 μ l during test, enzyme-linked immunosorbent assay instrument measures 450nm absorbance.Experiment in triplicate.
Result: as shown in Figure 1, wherein A is blank group result, and B is UVA irradiation group result, and C is 10-HDA group result, the absorbance wherein setting blank group represents the cytoactive of 100%, and the absorbance ratio of the absorbance that B, C respectively organize and A group represents the Cell relative activity of each group; Wherein white post represents that the testing result after cultivating 24h, black post represent the testing result after cultivating 48h.Compared with blank group, the cell viability of UVA irradiation group declines, and prompting UVA irradiates and produces toxicity to cell, and higher with its vigor of cell of 10-HDA process, the cytotoxicity that prompting 10-HDA can suppress UVA to induce.After 10-HDA process 48h, the cytoactive of 10-HDA group obviously increases, and difference has statistical significance (P < 0.05) compared with UVA irradiation group.
(4) active oxygen (ROS) level determination
Experimental technique: experiment is divided into 3 groups: blank group, UVA irradiation group and 10-HDA group.Blank group: normal culture fluid is cultivated; UVA irradiation group: use 4J/cm
2uVA irradiates, and the normal culture fluid of pre-irradiation is cultivated; 10-HDA group: be subdivided into 3 concentration groups, UVA pre-irradiation adds 10-HDA(0.5mmol/L, 1mmol/L, 1.5mmol/L of variable concentrations respectively) act on 24h, then use 4J/cm
2uVA irradiates.Be inoculated in by cell in 12 orifice plates, change liquid after cultivating 24h, 10-HDA group adds the culture fluid containing 10-HDA, and blank group and UVA irradiation group are then cultivated with normal culture fluid.4J/cm is given after cultivating 24h
2uVA irradiates, and has irradiated and has added 1ml immediately containing 10 μm of ol/LH
2the solution (the PBS solution Fresh with preheating) of DCFDA fluorescent probe, 37 DEG C of lucifuges hatch 30min, have hatched the PBS solution adding 1ml preheating and have washed 2 times to remove residual H
2dCFDA, add and do not digest 3min containing the pancreatin 37 DEG C of phenol red 0.25%, basis of microscopic observation to cell retraction, come off time, add immediately containing 10%FBS but do not stop digestion containing phenol red DMEM culture fluid, cell suspension is transferred in opaque 96 well culture plates, in fluorescence microplate reader fluorescence intensity, excitation wavelength is 490nm, and emission wavelength is 510-570nm.
Result: as shown in Figure 2, the 10-HDA that wherein A is blank group, B is UVA irradiation group, C, D, E are respectively 0.5mmol/L, 1mmol/L, 1.5mmol/L concentration in 10-HDA group.The intracellular ROS level of UVA irradiation group is about 1.4 times of blank group, and both comparing differences have height statistical significance (P<0.01), and prompting UVA irradiates and can produce ROS in induced fibroblast.Can decline in dose dependent by intracellular ROS level after the 10-HDA process of 0.5mmol/L-1.5mmol/L, difference has statistical significance (P<0.05) compared with UVA irradiation group, the ROS that UVA can be suppressed to induce after prompting 10-HDA process cell.
(5) cell ageing beta galactosidase (SA-β-gal) dyeing
Experimental technique: experiment is divided into 3 groups: blank group, UVA irradiation group and 10-HDA group.Blank group: normal culture fluid is cultivated; UVA irradiation group: use 4J/cm
2uVA irradiates, and changes normal culture fluid and cultivate after irradiation; 10-HDA group: be subdivided into 3 concentration groups, use 4J/cm
2uVA irradiates, and adds 10-HDA(0.5mmol/L, 1mmol/L, 1.5mmol/L of variable concentrations after irradiation respectively).Cell is inoculated in 12 orifice plates, cultivates 24h, give 4J/cm
2uVA irradiates, and adopts containing 10-HDA or do not dye containing carrying out SA-β-gal after the culture fluid cultivation 24h of 10-HDA after irradiation.Staining procedure is as follows: inhale and abandon culture medium, PBS solution washes 1 time, and the fixative room temperature adding 500 μ l pre-assigned fixes 15min.The PBS solution of fixing rear 1ml washes 2 times, then adds the dye liquor that 500 μ l are pre-assigned, hatches 24-36h for 37 DEG C.Beta galactosidase staining positive cells rate computational methods: often group chooses four visuals field continuously under 200 × mirror, positive cell is blue, i.e. senile cell, positive cell rate=positive cell number/total cellular score × 100%.
Result: as shown in Figure 3, the 10-HDA that wherein A is blank group, B is UVA irradiation group, C, D, E are respectively 0.5mmol/L, 1mmol/L, 1.5mmol/L concentration in 10-HDA group.Compared with blank group, UVA irradiation group SA-β-gal staining positive cells obviously increases, and difference has height statistical significance (P<0.01), and prompting UVA irradiates can induced fibroblast aging.As can be seen from the figure, after cell 0.5mmol/L10-HDA process, SA-β-gal staining positive cells has decline compared with UVA irradiation group, statistical analysis shows, after 1mmol/L10-HDA process, the decline compared with UVA irradiation group of SA-β-gal staining positive cells has statistical significance, and after 1.5mmol/L10-HDA process, SA-β-gal staining positive cells declines significantly compared with UVA irradiation group.Cell is that dose dependent declines by the quantity of cell ageing after (0.5-1.5mmol/L) 10-HDA process, the cell ageing that UVA can be suppressed to induce after prompting 10-HDA process cell.
(6) collagen content measures
Experimental technique: experiment is divided into two large groups, and first group irradiates without UVA, second largest group gives UVA and irradiates.Each large group is all subdivided into 2 groups: matched group and 10-HDA group.Matched group: normal culture fluid is cultivated; 10-HDA group: containing 1mmol/L10-HDA in culture fluid.Fibroblast is inoculated in 6 orifice plates, after cultivating 24h, uses 4J/cm for second largest group
2uVA irradiates, and uses the collagen content detecting supernatant after the fresh culture containing or do not contain 10-HDA continues to cultivate 48h after irradiation instead.First group except without UVA irradiate, other operation with second largest group.The detailed step that collagen content measures is as follows: collect cell conditioned medium liquid, low-speed centrifugal (500g, 5min) 1 time, then high speed centrifugation (12000rpm, 10min) 1 time is to remove impurity.Get 800 μ l centrifugal after supernatant be transferred in the centrifuge tube of low protein adheres, add the collagen protein isolation and identification reagent of 160 μ l, centrifuge tube put into the container containing mixture of ice and water, 4 DEG C of overnight incubation.Hatch rear careful taking-up and centrifugal (12000rpm, 10min), careful suction abandons supernatant, add 800 μ lSircol dye liquors, vortex oscillation mixes, and on shaking table, ambient temperature with gentle rocks 30min, centrifugal (12000rpm, 10min), supernatant is abandoned in careful suction, adds the base reagent of 250 μ l, and vortex oscillation mixes, get after mixing in 200 μ l mixed liquor to 96 hole ELISA Plate, enzyme-linked immunosorbent assay instrument measures 570nm absorbance.Often group establishes two multiple holes, and experiment in triplicate.
Result: as shown in Figure 4, wherein A matched group, B are 10-HDA group, and white post represents and to irradiate without UVA, and black post represents and to irradiate through UVA.Adopt the soluble collagen in Sircol collagen assay kit detection cell supernatant.Cell measures after after UVA irradiates, the culture fluid added containing 10-HDA hatches 48h.Compared with blank group, the collagen content of cell in supernatant after 1mmol/L10-HDA process increases.Compared with blank group, the collagen content of UVA irradiation group increases, and prompting UVA can increase the collagen content in Fibroblast culture solution.Compared with UVA irradiation group, the collagen content in 10-HDA group supernatant increases.
(7) quantitative fluorescent PCR
Experimental technique: the level adopting fluorescence quantitative PCR detection Fibroblast collagenase (MMP-1) and MMP-3mRNA.Experiment is divided into two large groups, and first group irradiates without UVA, and second largest group gives UVA and irradiate.Each large group is all subdivided into 2 groups: matched group and 10-HDA group.Matched group: normal culture fluid is cultivated; 10-HDA group: containing 1mmol/L10-HDA in culture fluid.Get 3-8 for fibroblast by 1 × 10
5the density of cells/well is inoculated in 6 orifice plates, and every hole adds the culture fluid of 2ml, gives 4J/cm after cultivating 24h
2uVA irradiates, and extracts cell total rna and synthesis cDNA after irradiating 24h, then adopts two-step method PCR response procedures to carry out quantitative fluorescent PCR reaction.Reaction terminates amplification curve and the melt curve analysis of rear confirmation quantitative fluorescent PCR, and take GAPDH as internal reference gene, interpretation of result adopts international 2-Δ Δ Ct method.
Cell total rna extracts the RNA extraction method adopting laboratory routine, RNA be stored in-80 DEG C for subsequent use.With the total cRNA of cell extracted for template adopts laboratory conventional method synthesis cDNA, the cDNA of synthesis be stored in-20 DEG C for subsequent use.
Quantitative fluorescent PCR reaction system:
premixExTaq
tMiI 10.0 μ l, 10 μMs of forward primer 0.8 μ l, 10 μMs of downstream primer 0.8 μ l, cDNA template 100ng, supplement sterilizing deionized water to 20 μ l.The wherein fluorescent dye that uses for conventional real-time fluorescence PCR of SYBR fluorescent dye, upstream and downstream primer is see table 1.
Reaction condition, 95 DEG C of denaturation 30sec, then enter 40 circulations: 95 DEG C of 5sec, 60 DEG C of 20sec.Melt curve analysis analysis is carried out after having reacted.
Table 1 fluorescent quantitative PCR primer
Result: as shown in Figure 5 and Figure 6, Fig. 5 is the testing result of MMP-1, and Fig. 6 is the testing result of MMP-3, and wherein, A is blank group, and B is 10-HDA group, and white post represents and to irradiate without UVA, and black post represents and to irradiate through UVA.Fluorescent quantitative PCR result shows, intracellular MMP-1 and the MMP-3mRNA level of UVA irradiation group is 3.7 times and 5.9 times of blank group respectively, all have height statistical significance (P<0.01) with blank group comparing difference, prompting UVA irradiates and can express MMP-1 and MMP-3mRNA by induced fibroblast.Compared with UVA irradiation group, cell MMP-1 and the MMP-3mRNA level of cell after 10-HDA process declines, the expression of MMP-1 and MMP-3mRNA that UVA can be suppressed to induce after prompting 10-HDA process cell.
(8) protein immunoblotting (Westernblot):
Experimental technique: adopt Westernblot to detect the level of MMP-1, MMP-3, JNK and p38MAPK albumen.Experiment is divided into two large groups, and first group irradiates without UVA, and second largest group gives UVA and irradiate.Each large group is all subdivided into 2 groups: matched group and 10-HDA group.Matched group: normal culture fluid is cultivated; 10-HDA group: containing 1mmol/L10-HDA in culture fluid.Get 3-8 for fibroblast by 1 × 10
5the density of cells/well is inoculated in 6 orifice plates, and every hole adds the culture fluid of 2ml, gives 4J/cm after cultivating 24h
2uVA irradiates.After UVA irradiates, cultivate 24h when detecting MMP-1 and MMP-3 protein level and extract total protein of cell, after UVA irradiates, then cultivate 1.5h when detecting JNK and p38MAPK protein level and extract total protein of cell.Often group collects the protein sample of equivalent, the protein sample of collection is loaded in SDS-PAGE glue well, then carries out electrophoresis, transferring film, close and hatch primary antibodie (mouse-anti people MMP-1 antibody, mouse-anti people MMP-3 antibody, rabbit anti-human p-JNK antibody, rabbit anti-human t-JNK antibody, rabbit anti-human p-p38MAPK antibody and the anti-human t-p38MAPK antibody of rabbit).Hatch two anti-after carry out develop the color (two of horseradish peroxidase-labeled resists).After colour developing, film is scanned, by the picture density of gel images processing system ImageJ software analysis band after scanning, make the relative expression quantity of correction calculation destination protein with reference gene β-actin.
Result: as is seen in figs 7-10, Fig. 7 is the testing result of MMP-1, and Fig. 8 is the testing result of MMP-3, and Fig. 9 is the testing result of JNK, and Figure 10 is the testing result of p38, is wherein I gel images, and II is quantitative analysis post figure; A is matched group, and B is 10-HDA group, and white post represents and to irradiate without UVA, and black post represents and to irradiate through UVA.Compared with blank group, intracellular MMP-1, MMP-3, p-JNK and p-p38MAPK protein level of UVA irradiation group obviously increases, and prompting UVA irradiates and can express the expression of MMP-1, MMP-3, p-JNK and p-p38MAPK albumen by induced fibroblast.The cell that UVA irradiates MMP-1, MMP-3, p-JNK and p-p38MAPK protein level after 10-HDA process declines, the expression of MMP-1, MMP-3, p-JNK and p-p38MAPK albumen that UVA can be suppressed to induce after prompting 10-HDA process cell.
In this example, the confirmatory experiment of above-mentioned (3) to (8) all repeats at least 3 times; And adopt SPSS13.0 statistical software to carry out variance analysis to each group of data obtained in experiment, more again with the LSD inspection in variance analysis between two groups.Experimental data represents with mean ± standard deviation (means ± SD).
The experimental result of above-mentioned (3) to (8) of this example shows, the cytotoxicity that 10-HDA can suppress UVA to induce, the generation suppressing the cell ageing of UVA induction, suppress UVA induction ROS, and increase collagen content, illustrate that 10-HDA has the effect of anti-light aging, its mechanism and the activation of suppression JNKandp38MAPK signal path and reduce MMP-1 and MMP-3 and express relevant.Therefore, 10-HDA is a kind of effective anti-skin photoage material.
Embodiment two is containing the anti-photoaging formulation of Royal Jelly acid (10-HDA)
10-HDA and the emulsifying agent such as stearic acid, liquid paraffin are mixed and made into 10-HDA emulsion.Select 24 health adults, emulsion containing 10-HDA or blank (not containing 10-HDA) are coated in two places inside right arm respectively, use emulsion every day three times, continuous use 15 days, experiment start use ultraviolet radiation arm, every three days photographs once, shine secondary altogether, within the 1st, 3,5,7,10,15 day, observe local erythema and pigmentation situation after irradiation respectively.
Result shows, after irradiation the 1st, 3,5, the 7 day all visible erythema of ultraviolet radiation place skin, and the erythema area at the 10-HDA emulsion place of smearing and color all comparatively the blank place of smearing are light.The pigmentation of the 10th, the 15 day all visible brown in ultraviolet radiation place after irradiation, the pigmentation area at the 10-HDA emulsion place of smearing and color all comparatively the blank place of smearing are light.Therefore the erythema that causes after can alleviating ultraviolet radiation skin of 10-HDA emulsion and pigmentation.Visible, life-time service is containing the clinical symptoms can improving skin photoage after the cosmetics of this composition of 10-HDA, play desalination wrinkle, reduce pigmentation, telangiectasis, increase skin elasticity, skin is compacted more, fine and smooth, and the dermopathic generations such as seborrheic keratosis, actinic granuloma, solar keratosis, malignant melanoma, basal cell carcinoma and squamous cell carcinoma can be prevented.
It should be noted that, it will be appreciated by those skilled in the art that, in view of the anti-skin photoage effect of 10-HDA, 10-HDA can add in skin care item, medicine cosmetic or other cosmetics by those skilled in the art easily, to prepare containing the skin care item with anti-light aging effect of 10-HDA, medicine cosmetic or other cosmetics.
In this example, 10-HDA is made emulsion, there is good anti-light aging effect.It will be understood by those skilled in the art that the dosage form of above-mentioned solvent, carrier or 10-HDA does not form the restriction to the application; Some compositionss containing 10-HDA can also be prepared, as long as said composition to be used in the beauty treatment of anti-skin photoage or treatment all in the protection domain of the application when not departing from the basic conception of the application.
Above content is the further description done the application in conjunction with concrete embodiment, can not assert that the concrete enforcement of the application is confined to these explanations.For the application person of an ordinary skill in the technical field, under the prerequisite not departing from the application's design, some simple deduction or replace can also be made, all should be considered as the protection domain belonging to the application.
Claims (1)
1. the application of anti-skin photoage preparation in anti-light aging skin care item, anti-light aging cosmetics or anti-light aging medicine cosmetic, is characterized in that: containing Royal Jelly acid in described anti-skin photoage preparation, the content of described Royal Jelly acid is 0.5-1.5mmol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210206686.0A CN103505374B (en) | 2012-06-20 | 2012-06-20 | A kind of anti-skin photoage preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210206686.0A CN103505374B (en) | 2012-06-20 | 2012-06-20 | A kind of anti-skin photoage preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103505374A CN103505374A (en) | 2014-01-15 |
| CN103505374B true CN103505374B (en) | 2016-02-10 |
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| CN106929231A (en) * | 2017-03-08 | 2017-07-07 | 蔡颖欣 | A kind of transparent amino acid crystal soap of Cinnamomum kanahirai hay and preparation method thereof |
| JP7232642B2 (en) * | 2018-12-28 | 2023-03-03 | ポーラ化成工業株式会社 | Anti-aging method and anti-aging agent |
| WO2020213743A1 (en) * | 2019-04-19 | 2020-10-22 | 株式会社 資生堂 | Method and device for preventing and/or improving photoaging and/or dermal pigmentation, anti-photoaging and/or dermal pigmentation inhibiting agent, method for screening anti-photoaging and/or dermal pigmentation inhibiting agent, method for evaluating anti-photoaging and/or dermal pigmentation inhibiting cosmetic treatment, and method for evaluating photoaging and/or dermal pigmentation |
| CN113144192A (en) * | 2020-01-22 | 2021-07-23 | 中国科学院上海营养与健康研究所 | Application of MAPK/ERK pathway inhibitor in antagonizing skin aging and radiation-induced skin premature aging |
| CN117653716B (en) * | 2023-12-08 | 2024-06-18 | 东莞胶原生物科技有限公司 | Application of succinyl end-lacking collagen capable of inhibiting iron death caused by skin photoaging |
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| CN1863526A (en) * | 2003-10-07 | 2006-11-15 | 株式会社林原生物化学研究所 | Collagen productin enhancer and production process and use thereof |
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