CN103505374A - Anti-skin photoaging preparation and application thereof - Google Patents
Anti-skin photoaging preparation and application thereof Download PDFInfo
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- CN103505374A CN103505374A CN201210206686.0A CN201210206686A CN103505374A CN 103505374 A CN103505374 A CN 103505374A CN 201210206686 A CN201210206686 A CN 201210206686A CN 103505374 A CN103505374 A CN 103505374A
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Abstract
The invention discloses an anti-skin photoaging preparation containing royal jelly acid (10-HDA), and also discloses application of 10-HDA (10-hydroxy-2-decenoic acid) in preparation of skin care products, cosmetics and cosmeceuticals. The anti-photoaging preparation provided by the invention can significantly improve the activity of UVA (ultra violet A) irradiated cells, inhibit UVA induction produced ROS (reactive oxygen species), inhibit UVA induced cell senescence, and has a significant anti-photoaging effect. Study on the action mechanism of the anti-skin photoaging preparation shows that the 10-HDA contained in the anti-photoaging preparation can inhibit activation of p-JNK and p38 MAPK signaling pathway and reduce UVA induced MMP-1 and MMP-3 expression, thereby increasing the collagen content in cells. Thus, the preparation is an effective anti-skin photoaging preparation.
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 adornment product.
Background technology
Skin aging mainly comprises intrinsic aging and exogenous aging.Exogenously agingly refer to that skin is subject to such environmental effects and the aging that causes changes, because take the impact of ultraviolet (Ultraviolet, UV) radiation as main, therefore be called again photoaging.Long wave ultraviolet (UVA) and ultraviolet B radiation (UVB) all can cause photoaging.Because UVB is strong 1000 times compared with UVA to the effect of burning of skin, UVB is considered to cause the main spectrum of photoaging always for a long time.Yet nearest research shows that UVA is only the main spectrum that causes skin photoage.
The main clinical manifestation of skin photoage is that wrinkle of skin, coarse, dry, lax, telangiectasis, fragility increase and pigmentation.Long-term sun exposure can cutaneous various kinds of cell composition and organizational structure change, wherein the distinctive variation of tool is the change of hypodermal cell epimatrix composition.Hypodermal cell epimatrix comprises collagen fiber, elastic fibers, aminopolysaccharide and proteoglycan etc., and wherein type i collagen albumen is its major structural protein.At photoaging skin, I type and III type collagen fiber reduce, disorder, and in bulk is assembled in elastic fiber degeneration, chap, and aminopolysaccharide cracking, causes the generation of cutis laxa, wrinkle.
The mechanism of skin photoage is not yet completely clear, but matrix metalloproteinase (Matrix metalloproteinases, MMPs) expression is increased or increased activity is the important mechanisms of the matrix components degradeds such as collagen fiber.MMPs Shi Yizu zinc protease family, by 25 member compositions such as MMP-1, MMP-2, MMP-3.According to being that soluble protein or membrane bound protein are divided into collagen enzyme, gelatin enzyme, stromelysin and membranous type MMPs after its substrate specificity and their secretion.In 19 kinds of MMPs of normal human skin expression, only have three kinds to UV, to irradiate and make a response and significantly induced, i.e. MMP-1, MMP-3 and MMP-9.UV irradiated after 24 hours, and MMP-1 and MMP-3 mRNA level significantly increase, and MMP-9 mRNA level only has appropriateness to increase.
The MMPs of UV radiation-induced plays an important role in the degradation process of skin photoage hypodermal cell epimatrix composition.After ultraviolet radiation skin, can induce the multiple matrix metalloproteinase proenzyme of keratinocyte and fibroblasts to secrete, and by regulating the activation of proenzyme and the expression of matrix metallo-proteinase inhibitor to make it change into activated MMPs.The hypodermal cell epimatrix composition that the MMPs of activation can degrade nearly all, causes that the skin photoagees such as cutis laxa, wrinkle formation change.
The dermal matrix albumen that dissimilar MMP degrades different.For example, MMP-1 degraded I, II and III collagen type, MMP-9 degrade IV, V collagen protein and gelatin, MMP-3 has substrate specificity widely, its can degrade polytype collagen protein and substrate molecule, as proteoglycan, laminin，LN and fibronectin.For collagen protein, especially I type and III collagen type, first start collagen fiber by MMP-1 and rupture, and then upborne MMP-3 and MMP-9 further degrade.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 that the skin photoagees such as cutis laxa, wrinkle formation 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 main cell source of MMPs.UV radiation-induced MMPs gene expression relates to complicated cell signal path, 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.The AP-1 of activation and NF-κ B can be respectively combined with the AP-1 of multiple MMPs promoter and NF-kB site and the expression of inducing MMPs.Verified, the AP-1 that UV activates stimulates transcribing of keratinocyte and fibroblast MMP-1 and MMP-3.Because MMP-1 has similar structure with MMP-3 promoter, UVB irradiates postactivated AP-1 is combined and causes transcribing of MMP-1 and MMP-3 with the AP-1 site of MMP-1 and MMP-3 promoter respectively.The generation of the active oxygen (Reactive oxygen species, ROS) that UV irradiation causes is considered to activate an initiating link of above-mentioned two signal paths.
In sum, UV irradiates can be by activating the activation of complicated signal path induction MMPs, and then degraded hypodermal cell epimatrix composition causes skin photoage, initiating link or the key link of therefore blocking these signal paths just can suppress the effect that MMPs activation reaches anti-light aging.In recent years, MMPs is considered to a promising anti-light aging treatment target spot.Research and develop effective MMPs inhibitor and become an important study hotspot.Up to the present, there is the studied exploitation of MMPs inhibitor that comes from a large number natural biological.They be take respectively ROS, MAPK path, NF-κ B path or a plurality of link and reach the effect of anti-light aging as target spot.The MMPs inhibitor that the ROS of take is target spot is as white floss Rhizoma Polypodiodis Nipponicae leaf extract, phylloxanthin, little coralline algae etc.These materials can suppress by removing ROS the expression of MMPs.The MMPs inhibitor that the MAPK path of take is target spot as fucoidan, trans zeatin etc. can be by blocking-up UV mediation MAPK path to suppress the expression of MMPs.The NF-κ B path that the MMPs inhibitor that the NF-κ B path of take is target spot can mediate by inhibition UV as cordycepin, magnolol etc. suppresses the expression of MMPs.
Royal Jelly acid (chemical name: 10-HAD, 10-hydroxy-2-decenoic acid, 10-HDA) is a kind of natural materials of the glandular secretions such as the 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, in nineteen twenty-one, found first.From then on, about the research of Royal Jelly acid, reported successively, disclose Royal Jelly acid and there is medical health effect, aspect acute radiation injury, there is 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 having relevant research report at present aspect the anti-light aging of Royal Jelly acid.
Summary of the invention
The application's object is to provide a kind of new anti-skin photoage preparation, and the application of 10-HDA in preparing anti-light aging skin care item, anti-light aging cosmetics, anti-light aging medicine adornment product.
To achieve these goals, the application has adopted following technical scheme:
The application discloses a kind of anti-skin photoage preparation, and 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 and in this anti-skin photoage preparation, also contain other pharmaceutically acceptable carrier, solvent, or also can contain typical additives of skin protection, skin Caring etc.
Further, in above-mentioned anti-light aging preparation, 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 adornment product.
Concrete, the application discloses a kind of anti-light aging skin care item, and 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 adornment product, the main active substances of its anti-light aging comprises Royal Jelly acid.
Further, in above-mentioned medicine adornment product, the content of Royal Jelly acid is 0.5-1.5mmol/L.
Owing to adopting above technical scheme, the application's beneficial effect is:
The application provides a kind of new anti-light aging preparation, wherein contains 10-HDA.Experimental result shows, 10-HDA can obviously improve the activity of the postradiation cell of UVA, suppresses UVA induction and produces ROS, suppresses the cell ageing of UVA induction, has the effect of significant anti-light aging.Studies show that, the 10-HDA containing in the application's anti-light aging preparation can suppress the activation of p-JNK and p38 MAPK signal path, reducing the MMP-1 of UVA induction and the expression of MMP-3, thereby increase the collagen content in cell, is a kind of preparation of effective anti-skin photoage.
Accompanying drawing explanation
Fig. 1 is the vitality test result of Royal Jelly acid to the postradiation cell of process UVA in the embodiment of the present application;
Fig. 2 is the affect result of Royal Jelly acid on active oxygen in the embodiment of the present application;
Fig. 3 is the anti-light aging effect detection of Royal Jelly acid in the embodiment of the present application;
Fig. 4 is the affect result of Royal Jelly acid on collagen protein in the embodiment of the present application;
Fig. 5 is the affect result of the Royal Jelly acid of fluorescence quantitative PCR detection in the embodiment of the present application on the mrna expression of MMP-1;
Fig. 6 is the affect result of the Royal Jelly acid of fluorescence quantitative PCR detection in the embodiment of the present application on the mrna expression of MMP-3;
Fig. 7 is Royal Jelly acid that in the embodiment of the present application, protein immunoblotting the detects result that affects on MMP-1 albumen;
Fig. 8 is Royal Jelly acid that in the embodiment of the present application, protein immunoblotting the detects result that affects on MMP-3 albumen;
Fig. 9 is Royal Jelly acid that in the embodiment of the present application, protein immunoblotting the detects result that affects on JNK albumen;
Figure 10 is Royal Jelly acid that in the embodiment of the present application, protein immunoblotting the detects result that affects on p38 albumen.
The specific embodiment
Present inventor has confirmed that by a series of experiment 10-HDA can suppress the cytotoxicity of UVA induction and the generation of ROS, suppress the fibroblast aging of UVA induction, the activation that suppresses p-JNK and p38MAPK signal path, the MMP-1 that reduces UVA induction and the expression of MMP-3, increases the collagen content in cell conditioned medium liquid.
On the basis of above-mentioned research, present inventor provides the new anti-skin photoage preparation of a kind of 10-HDA of containing.This anti-skin photoage preparation adds 10-HDA in medically acceptable solvent carrier (as liquid paraffin etc.) to and is prepared from, for the anti-light aging of skin.
Further, it will be appreciated by those skilled in the art that, anti-light aging effect in view of 10-HDA, can easily 10-HDA or above-mentioned anti-skin photoage preparation be added in skin care item, medicine adornment product or other cosmetics, to prepare the skin care item with anti-light aging effect, medicine adornment product or other cosmetics containing 10-HDA.
In addition, 10-HDA, except the skin care item for above-mentioned, cosmetics and medicine adornment product, 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 carrier that is applicable to locally apply on skin are prepared into the compositions with anti-light aging effect.
Below by specific embodiment, also by reference to the accompanying drawings the application is described in further detail.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 mono-Royal Jelly acid (10-HDA)
(1) human skin fibroblast is cultivated
Experimental technique: get child's postoperative prepuce tissues of peritomizing, the separated human fibroblasts that obtains purification, in the DMEM culture fluid of the superfine hyclone (FBS) containing 10% and mycillin, is placed in cell culture containing 5%CO
237 ℃ of incubators in cultivate.
(2) UVA irradiates
Experimental technique: cover with and give UVA while converging to 80%～90% and irradiate when fibroblast.Cell is taken out from incubator, inhale and abandon culture medium, with the phosphate buffer (PBS) of 37 ℃ of preheatings, wash 2 times.Every hole adds the thin layer PBS of equivalent, then with the UVA of various dose, irradiates.After irradiation, inhale and abandon PBS solution immediately, add freshly prepared normal culture fluid, be placed in 95% humidity, 5%CO
237 ℃ of incubators in cultivate.In this example, normal culture fluid is containing 10% superfine hyclone (FBS) and the DMEM culture fluid of mycillin.
(3) cell viability is measured
Experimental technique: whether can suppress the cytotoxicity of UVA induction in order to detect 10-HDA, this example is measured cell viability with CCK-8 reagent (Dojindo, Japan).Experiment is divided into 3 groups: blank group, UVA irradiation group and 10-HDA group, establish 3 Ge Fu holes for every 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: use 4J/cm
2uVA irradiates, and after irradiating, with the culture fluid containing 1mmol/L 10-HDA, cultivates.Fibroblast is pressed to 5 * 10
3cells/well density is inoculated in 96 well culture plate Zhong，Mei holes and adds 100 μ l culture fluid, and after inoculation 24h, UVA irradiation group and 10-HDA cell give respectively 4J/cm
2uVA irradiates, after irradiation, each organize cell according to above-mentioned condition add containing or not containing the culture fluid of 10-HDA, choose respectively after cultivating 24h, 48h and test.In test Shi Mei hole, add the CCK-8 reagent of 10 μ l, on enzyme-linked immunosorbent assay instrument, measure 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 of wherein setting blank group represents 100% cytoactive, the Cell relative activity of each group of dulling luminosity ratio value representation of the absorbance of each group of B, C and A group; Wherein white post represents to cultivate the testing result after 24h, and black post represents to cultivate the testing result after 48h.Compare with blank group, the cell viability of UVA irradiation group declines, and prompting UVA irradiates cell is produced to toxicity, and its vigor of cell of processing with 10-HDA is higher, and prompting 10-HDA can suppress the cytotoxicity of UVA induction.10-HDA processes after 48h, and the cytoactive of 10-HDA group obviously increases, and comparing difference with UVA irradiation group has statistical significance (P < 0.05).
(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 pre-irradiation is cultivated with normal culture fluid; 10-HDA group: be subdivided into 3 concentration groups, UVA pre-irradiation adds respectively 10-HDA(0.5mmol/L, 1mmol/L, the 1.5mmol/L of variable concentrations) effect 24h, then use 4J/cm
2uVA irradiates.Cell is inoculated in 12 orifice plates, after cultivation 24h, changes liquid, 10-HDA group is added the culture fluid containing 10-HDA, and blank group and UVA irradiation group are cultivated with normal culture fluid.After cultivating 24h, give 4J/cm
2uVA irradiates, and has irradiated and has added immediately 1ml to contain 10 μ mol/L H
2the solution of DCFDA fluorescent probe (with the fresh preparation of PBS solution of preheating), 37 ℃ of lucifuges are hatched 30min, have hatched and have added the PBS solution of 1ml preheating to wash 2 times to remove residual H
2dCFDA, add not the 37 ℃ of digestion 3min of pancreatin containing phenol red 0.25%, under microscope, observe cell retraction, while coming off, add immediately containing 10%FBS but containing phenol red DMEM culture fluid, do not stop digestion, 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, wherein A is that blank group, B are the 10-HDA that UVA irradiation group, C, D, E are respectively 0.5mmol/L, 1mmol/L in 10-HDA group, 1.5mmol/L concentration.1.4 times of left and right that intracellular ROS level is blank group are organized in UVA irradiation, and both comparing differences have height statistical significance (P < 0.01), and prompting UVA irradiates and can induce generation ROS in fibroblast.By intracellular ROS level after the 10-HDA processing of 0.5mmol/L-1.5mmol/L, can be dose dependent and decline, compare difference have statistical significance (P < 0.05) with UVA irradiation group, prompting 10-HDA can suppress the ROS that UVA induces after processing 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 respectively 10-HDA(0.5mmol/L, 1mmol/L, the 1.5mmol/L of variable concentrations after irradiation).Cell is inoculated in 12 orifice plates, cultivates 24h, give 4J/cm
2uVA irradiates, and adopts containing 10-HDA or after not cultivating 24h containing the culture fluid of 10-HDA and carry out SA-β-gal dyeing after irradiating.Staining procedure is as follows: inhale and abandon culture medium, PBS solution is washed 1 time, adds fixedly 15min of the pre-assigned fixative room temperature of 500 μ l.After fixing, with the PBS solution of 1ml, wash 2 times, then add the pre-assigned dye liquor of 500 μ l, hatch 24-36h for 37 ℃.Beta galactosidase stained positive cell rate computational methods: choose continuously the Si Ge visual field for every group 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, wherein A is that blank group, B are the 10-HDA that UVA irradiation group, C, D, E are respectively 0.5mmol/L, 1mmol/L in 10-HDA group, 1.5mmol/L concentration.Compare with blank group, UVA irradiation group SA-β-gal stained positive cell obviously increases, and difference has height statistical significance (P < 0.01), and prompting UVA irradiates and can induce fibroblast old and feeble.As can be seen from the figure, cell is processed rear SA-β-gal stained positive cell with 0.5mmol/L 10-HDA and has been compared decline with UVA irradiation group, statistical analysis shows, after 1mmol/L 10-HDA processes, SA-β-gal stained positive cell is compared with UVA irradiation group to decline and is had statistical significance, and after 1.5mmol/L 10-HDA processes, SA-β-gal stained positive cell is compared with UVA irradiation group and declined significantly.Cell is dose dependent decline by the quantity of cell ageing after (0.5-1.5mmol/L) 10-HDA processing, and prompting 10-HDA can suppress the cell ageing that UVA induces after processing cell.
(6) collagen content is measured
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: contain 1mmol/L 10-HDA in culture fluid.Fibroblast is inoculated in 6 orifice plates, after cultivation 24h, uses 4J/cm for second largest group
2uVA irradiates, and uses instead containing or containing the fresh culture of 10-HDA, do not continue to cultivate the collagen content that detects supernatant after 48h after irradiation.First group except irradiating without UVA, and other operate with second largest group.The detailed step that collagen content is measured is as follows: collect cell conditioned medium liquid, and low-speed centrifugal (500g, 5min) 1 time, then high speed centrifugation (12000rpm, 10min) 1 time is to remove impurity.Get the supernatant of 800 μ l after centrifugal and be transferred in the centrifuge tube that low albumen adheres to, add collagen protein separation and the concentrated reagent of 160 μ l, centrifuge tube is put into the container containing mixture of ice and water, 4 ℃ of overnight incubation.Hatch rear careful taking-up centrifugal (12000rpm, 10min), careful suction abandoned supernatant, add 800 μ l Sircol dye liquors, vortex oscillation mixes, and on shaking table, room temperature is jiggled 30min, centrifugal (12000rpm, 10min), careful suction abandoned supernatant, adds the base reagent of 250 μ l, and vortex oscillation mixes, after mixing, get in 200 μ l mixed liquor Zhi96 hole ELISA Plate, on enzyme-linked immunosorbent assay instrument, measure 570nm absorbance.Every group of She Lianggefu hole, experiment in triplicate.
Result: as shown in Figure 4, wherein A matched group, B are 10-HDA group, white post represents to irradiate without UVA, and black post represents to irradiate through UVA.Adopt Sircol collagen protein assay kit to detect the soluble collagen in cell conditioned medium liquid.Cell is measured after adding the culture fluid that contains 10-HDA to hatch 48h after UVA irradiates.Compare with blank group, cell increases with the collagen content that 1mmol/L 10-HDA processes in rear supernatant.Compare with blank group, the collagen content of UVA irradiation group increases, and prompting UVA can increase the collagen content in fibroblast culture fluid.Compare with UVA irradiation group, the collagen content in 10-HDA group supernatant increases.
(7) quantitative fluorescent PCR
Experimental technique: the level that adopts 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: contain 1mmol/L 10-HDA in culture fluid.Get 3-8 for fibroblast by 1 * 10
5the density of cells/well is inoculated in the culture fluid that 6 orifice plate Zhong，Mei holes add 2ml, after cultivation 24h, gives 4J/cm
2uVA irradiates, and after irradiation 24h, extracts cell total rna and synthetic cDNA, then adopts two-step method PCR response procedures to carry out quantitative fluorescent PCR reaction.Reaction finishes amplification curve and the melt curve analysis of rear confirmation quantitative fluorescent PCR, take GAPDH as internal reference gene, and interpretation of result adopts international 2-Δ Δ Ct method.
Cell total rna extract to adopt the RNA extracting method of laboratory routine, RNA be stored in-80 ℃ standby.The total cRNA of cell extracting of take is that template adopts the synthetic cDNA of laboratory conventional method, synthetic cDNA be stored in-20 ℃ standby.
Quantitative fluorescent PCR reaction system:
premix Ex Taq
tMiI 10.0 μ l, 10 μ M forward primer 0.8 μ l, 10 μ M downstream primer 0.8 μ l, cDNA template 100ng, supplement sterilizing deionized water to 20 μ l.Wherein SYBR fluorescent dye is the fluorescent dye that conventional real-time fluorescence PCR is used, and upstream and downstream primer is referring to table 1.
Reaction condition, 95 ℃ of denaturation 30sec, then enter 40 circulations: 95 ℃ of 5sec, 60 ℃ of 20sec.After having reacted, carry out melt curve analysis analysis.
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, wherein, A is blank group, and B is 10-HDA group, and white post represents through UVA, not irradiate, and black post represents to irradiate through UVA.Fluorescent quantitative PCR result shows, it is respectively 3.7 times and 5.9 times of blank group that intracellular MMP-1 and MMP-3 mRNA level are organized in UVA irradiation, all have height statistical significance (P < 0.01) with blank group comparing difference, prompting UVA irradiates and can induce fibroblast to express MMP-1 and MMP-3 mRNA.Compare with UVA irradiation group, cell declines by MMP-1 and MMP-3 mRNA level that 10-HDA processes rear cell, and prompting 10-HDA can suppress the MMP-1 of UVA induction and the expression of MMP-3 mRNA after processing cell.
(8) protein immunoblotting (Western blot):
Experimental technique: adopt Western blot to detect the level of MMP-1, MMP-3, JNK and p38 MAPK 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: contain 1mmol/L 10-HDA in culture fluid.Get 3-8 for fibroblast by 1 * 10
5the density of cells/well is inoculated in the culture fluid that 6 orifice plate Zhong，Mei holes add 2ml, after cultivation 24h, gives 4J/cm
2uVA irradiates.While detecting MMP-1 and MMP-3 protein level, after UVA irradiates, cultivate 24h and extract total protein of cell, while detecting JNK and p38 MAPK protein level, after UVA irradiates, cultivate 1.5h and extract total protein of cell.Every group of protein sample of collecting equivalent, the protein sample of collection is loaded in SDS-PAGE glue well, then carries out electrophoresis, transferring film, seal and hatch primary antibodie (mouse-anti people MMP-1 antibody, mouse-anti people MMP-3 antibody, the anti-human p-JNK antibody of rabbit, the anti-human t-JNK antibody of rabbit, rabbit anti-human p-p38 MAPK antibody and the anti-human t-p38 MAPK of rabbit antibody).Hatch and two develop the color after anti-(horseradish peroxidase-labeled two anti-).After colour developing, film is scanned, the picture after scanning is used to the density of gel images processing system ImageJ software analysis band, with reference gene β-actin, make the relative expression quantity of correction calculation destination protein.
Result: as shown in Fig. 7-10, the testing result that Fig. 7 is MMP-1, the testing result that Fig. 8 is MMP-3, the testing result that Fig. 9 is JNK, the testing result that Figure 10 is p38, is wherein I gel images, II is quantitative analysis post figure; A is matched group, and B is 10-HDA group, and white post represents to irradiate without UVA, and black post represents to irradiate through UVA.Compare with blank group, intracellular MMP-1, MMP-3, p-JNK and p-p38 MAPK protein level are organized in UVA irradiation obviously to be increased, and prompting UVA irradiates and can induce fibroblast to express the expression of MMP-1, MMP-3, p-JNK and p-p38 MAPK albumen.For the cell of UVA irradiation, 10-HDA processes rear MMP-1, MMP-3, p-JNK and the decline of p-p38 MAPK protein level, can suppress the expression of MMP-1, MMP-3, p-JNK and the p-p38 MAPK albumen of UVA induction after prompting 10-HDA processing 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 the data of respectively organizing that obtain in experiment, between two groups, with the LSD in variance analysis, check more again.Experimental data represents with mean ± standard deviation (means ± SD).
The experimental result of above-mentioned (3) to (8) of this example shows, 10-HDA can suppress the generation of the cytotoxicity of UVA induction, the cell ageing that suppresses UVA induction, inhibition UVA induction ROS, and increase collagen content, illustrate that 10-HDA has the effect of anti-light aging, its mechanism reduces MMP-1 with suppressing the activation of JNK and p38 MAPK signal path and MMP-3 expresses relevant.Therefore, 10-HDA is a kind of effective anti-skin photoage material.
Embodiment bis-is containing the anti-light aging preparation of Royal Jelly acid (10-HDA)
The emulsifying agents such as 10-HDA and stearic acid, liquid paraffin are mixed and made into 10-HDA emulsion.Select 24Ge health adult, the emulsion that contains 10-HDA or blank (containing 10-HDA) are coated in respectively to two places of right arm inner side, use emulsion every day three times, use 15 days continuously, in experiment, start to use ultraviolet radiation arm, every three days photographs once, shine altogether secondary, within the 1st, 3,5,7,10,15 days after irradiation, observe respectively local erythema and pigmentation situation.
Result shows, the equal visible erythema of the 1st, 3,5,7 days ultraviolet radiation place skin after irradiation, and the erythema area at the 10-HDA emulsion place of smearing and color are all compared with the blank place of smearing gently.The equal pigmentation of visible brown in the 10th, 15 days ultraviolet radiation places after irradiation, the pigmentation area at the 10-HDA emulsion place of smearing and color are all compared with the blank place of smearing gently.Therefore 10-HDA emulsion can alleviate erythema and the pigmentation causing after ultraviolet radiation skin.Visible, life-time service is containing the clinical symptoms that can improve skin photoage after the cosmetics of this composition of 10-HDA, play desalination wrinkle, reduce pigmentation, telangiectasis, increase skin elasticity, make that skin compacts more, exquisiteness, and can prevent the dermopathic generations such as seborrheic keratosis, actinic granuloma, solar keratosis, malignant melanoma, basal cell carcinoma and squamous cell carcinoma.
It should be noted that, it will be appreciated by those skilled in the art that, anti-skin photoage effect in view of 10-HDA, those skilled in the art can add 10-HDA in skin care item, medicine adornment product or other cosmetics easily, to prepare the skin care item with anti-light aging effect, medicine adornment product or other cosmetics containing 10-HDA.
In this example, 10-HDA is made to emulsion, there is good anti-light aging effect.The dosage form that it will be understood by those skilled in the art that above-mentioned solvent, carrier or 10-HDA does not form the restriction to the application; In the situation that do not depart from the application's basic conception, can also prepare some compositionss containing 10-HDA, as long as said composition is used in the beauty treatment of anti-skin photoage or treatment all in the application's protection domain.
Above content is the further description of the application being done in conjunction with concrete embodiment, can not assert that the application's concrete enforcement is confined to these explanations.For the application person of an ordinary skill in the technical field, not departing under the prerequisite of the application's design, can also make some simple deduction or replace, all should be considered as belonging to the application's protection domain.
Claims (9)
1. an anti-skin photoage preparation, is characterized in that: contain Royal Jelly acid.
2. anti-skin photoage preparation according to claim 1, is characterized in that: the content of described Royal Jelly acid is 0.5-1.5mmol/L.
3. the application of anti-skin photoage preparation according to claim 1 and 2 in anti-light aging skin care item, anti-light aging cosmetics or anti-light aging medicine adornment product.
4. anti-light aging skin care item, is characterized in that: contain Royal Jelly acid.
5. anti-light aging skin care item according to claim 4, is characterized in that: the content of described Royal Jelly acid is 0.5-1.5mmol/L.
6. anti-light aging cosmetics, is characterized in that: contain Royal Jelly acid.
7. anti-light aging cosmetics according to claim 6, is characterized in that: the content of described Royal Jelly acid is 0.5-1.5mmol/L.
8. anti-light aging medicine adornment product, is characterized in that: contain Royal Jelly acid.
9. anti-light aging medicine adornment product according to claim 8, is characterized in that: the content of described Royal Jelly acid is 0.5-1.5mmol/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| JP2020105125A (en) * | 2018-12-28 | 2020-07-09 | ポーラ化成工業株式会社 | Anti-aging method and anti-aging agent |
| 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 |
| CN113784753A (en) * | 2019-04-19 | 2021-12-10 | 株式会社资生堂 | Method and device for preventing and/or ameliorating photoaging and/or dermal pigmentation, photoaging-resistant and/or dermal pigmentation inhibitor, screening method for photoaging-resistant and/or dermal pigmentation inhibitor, evaluation method for photoaging-resistant and/or dermal pigmentation-inhibiting cosmetic treatment, and evaluation method for photoaging-resistant and/or dermal pigmentation degree |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| JP2020105125A (en) * | 2018-12-28 | 2020-07-09 | ポーラ化成工業株式会社 | Anti-aging method and anti-aging agent |
| 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 |
| CN113784753A (en) * | 2019-04-19 | 2021-12-10 | 株式会社资生堂 | Method and device for preventing and/or ameliorating photoaging and/or dermal pigmentation, photoaging-resistant and/or dermal pigmentation inhibitor, screening method for photoaging-resistant and/or dermal pigmentation inhibitor, evaluation method for photoaging-resistant and/or dermal pigmentation-inhibiting cosmetic treatment, and evaluation method for photoaging-resistant and/or dermal pigmentation degree |
| 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 |
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| Publication number | Publication date |
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| CN103505374B (en) | 2016-02-10 |
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