鱼鳞胶原蛋白肽的大小和结构对脸部皮肤质量的作用和穿

鱼鳞胶原蛋白肽的大小和结构对脸部皮肤质量的作用和穿 Journal of Biomedicine and BiotechnologyVolume 2010 (2010), Article ID 757301, 9 pagesdoi:ResearchArticleEffectsof Sizes and Conformations of Fish-Scale Collagen Peptides onFacial Skin Qualities and Transdermal PenetrationEfficiencyHuey-Jine Chai,1,2 Jing-Hua Li,2 Han-Ning Huang,1 Tsung-Lin Li,3Yi-Lin Chan,4 Chyuan-Yuan Shiau,1 and Chang-Jer Wu11Department of Food Science, National Taiwan Ocean University,Keelung 20224, Taiwan2Seafood Technology Division, Fisheries Research Institute,Council of Agriculture, Keelung 20246, Taiwan3Genomics Research Center, Academia Sinica, Taipei11529,Taiwan4Institute of Biomedical Sciences, Academia Sinica, Taipei115,TaiwanReceived 25 February 2010; Accepted 3 April 2010Academic Editor: Rumiana KoynovaCopyright © 2010 Huey-Jine Chai et al. This is an open accessarticle distributed under the Creative Commons Attributio?,,sifu www.kakawgw.com dongdingn License,which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.AbstractFish-scale collagen peptides (FSCPs) were prepared using a givencombination of proteases to hydrolyze tilapia (Oreochromis sp.)scales. FSCPs were determined to stimulate fibroblast cellsproliferation and procollagen synthesis in a time- anddose-dependent manner. The transdermal penetration capabilities ofthe fractionationed FSCPs were evaluated using the Franz-typediffusion cell model. The heavier FSCPs, 3500 and 4500 Da,showed higher cumulative penetration capability as opposed to thelighter FSCPs, 2000 and 1300 Da. In addition, the heavierseemed to preserve favorable coiled structures comparing to thelighter that presents mainly as linear under confocal scanninglaser microscopy. FSCPs, particularly theheavier, were concluded to efficiently penetrate stratum corneum toepidermis and dermis, activate fibroblasts, and accelerate collagensynthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preservingthe given desired structure feature.1.IntroductionCollagens are the major structural element of connective tissuesin vertebrate, comprising 30% or so of total protein. They also exist in theinterstitial tissues of virtually all parenchymal organs, whereinthey stabilize organs and keep them in good shapes [1]. To date,over 20 types of collagens have been identified, and they wereotherwise divided into three groups, fibrous collagen, fibrilassociated collagen, and basement membrane collagen[1–3].Skin, the largest organ in human body protects the body from variedexternal insults. It is composed of three layers, namely theepidermis, the dermis, and the subcutaneous layer. Each layerprovides some extents of physical strength and flexibility inconcert with other physiological functions [4]. The dermis containsconsiderable amounts of extracellular matrix (ECM), such ascollagens and glycosaminoglycans (GAGs) produced mainly byfibroblasts. Collagen type I [5] known to maintain the integral ofthe dermis is the most abundant collagen in human body. Aging skinis admittedly related to the reduction of collagen type I [6].Collagen has been widely processed as products in food,cosmetic, biomedical, and pharmaceutical industries. Oralconsumption of collagen peptides may provide some beneficialeffects for the body. For examples, collagen peptides as a foodsupplement may improve low bone mineral density in people inmalnutrition and people suffering degenerative joint diseases[7–9]. Reports also indicated that consumption of collagen canthicken hairs [10], improve nail disorders such as brittle nails[11], increase the size of collagen fibrils in Achilles tendon[12], induce fibroblast density, and enhance formation of collagenfibrils in dermis etcetera [13]. Traditionally, collagens arederived from livestock sources, such as bovine hide and bones aswell as swine skin [14]. Because of the outbreaks of bovinespongiform encephalopathy (BSE) and foot-and-mouth disease (FMD) inrecent years, collagens and collagen-derived products from suchsources may have intimidated considerable users [15]. Collagens andcollagen-derived products from swine sources have an additionaldisadvantage as religious sake in some parts of the world [16].,,Fish wastes,, therefore turn out to be a good alternative. In fishprocessing, a large portion of wastes would generate, whichtypically accounts for 50%–70% of raw materials including skins,bones, scales, viscera, and heads [17]. These so-called ,,wastes,,were difficult to handle, generally used as low-value feedstuffs orfertilizers as a whole, so that serious environmental issues arisefrom time to time [18]. Given 30% or so of collagen contained inthese ,,wastes,,, they have drawn great attention lately [19].The stratum corneum (SC) has been known to minimize passivewater loss from the body, to ward off absorption of chemicals inthe environment, and to prevent microbial invasion [19, 20].Topical administration of cosmetics or biomedical materials on skintherefore has to overcome the stratum corneum barrier so as toreach the fibroblast cells in the dermal layer. Raw collagensderived from whatever sources without any process for eithercosmetic or biomedical purposes may not have desirable effects, forinstance the type I collagen proliferation. We considered factorssuch as molecular volume (MV) or molecular weight (MW) of collagensmay play a key role on their penetration ability when administratedtopically [21], of which relevant reports remained few.In this paper, we prepared testing collagens using scale wastesof tilapia (Oreochromis sp), which is a high value-added fish inTaiwan fishery. The prepared collagens formulated into skin essencewere assessed for their physiological effects on facial skin inusers. In addition, we determined the levels of fibroblastsproliferation and collagen synthesis in embryonic fibroblast celllines after being subjected to FSCP treatments. Given molecularsizes of FSCPs on transdermal efficiencies were also explored. Infact, our results showed that the transdermal penetrationefficiency of FSCPs is positively correlated to given FSCPs,,molecular sizes and structural features in a nude mice model.2. Materials and Methods. Preparation of Fish Scale Collagen Peptides (FSCPs)Fishscale collagen peptides (FSCPs) were isolated from tilapia(Oreochromis sp.) scales (Ko-Fwu Fishes Co., Taiwan) by enzymatichydrolysis according to our patented protocol [22]. In short, fishscales were washed to rid of impurities and then heated for 15minutes at 121,, C to soften scales. The heated scales(200 g) were smashed into small pieces by disperser(Kinematica, NY, USA), then subjected to hydrolysis under 1%Protease N for hours and % Flavourzyme (Novozymes,Chiba-shi, Japan) for another hour at an optimal pH andtemperature. Hydrolysates were stirred and heated in a boilingwater bath for 10 min to inactivate enzymes. Then, thehydrolysates were centrifuged at 12,000 g for 20 min. Thesupernatants (FSCPs) were lyophilized and stored at -20º C foruse.. Moisture Contents and Relative Elasticity AssaysFSCP-based skin essences were formulated into 5%, 7%, and 10% oftotal FSCPs as the common basal ingredients. Sixty-two voluntaryTaiwanese women (within 23 to 60 years of age) were subjected tothe FSCP-based skin essence treatment on facial skin twice a dayfor 30 days. These test subjects were forbidden using any othercosmetic products during the test. Moisture contents and relativelyelasticity of facial skins were measured every other week (at the0, 2nd, and 4th week) by the skin probe of Cutometer MAP 580 (KOKO,Leichlingen, Germany) at 20–22ºC and at 40%–60% relative humidity.Each measurement took place at 30 min after washing face withDI water.. Cell Proliferation and Collagen Release AssaysAbout 2 x 105 Detroit 551 cells (human embryonic skinfibroblast cell line, ATCC CCL-110) and STO cells (mouse embryonicfibroblast cell line, ATCC CRL-1503) were seeded into each well ofa 96-well plate and maintained in eagle,,s minimum essential medium(EMEM) and Dulbecco,,s modified Eagle,,s medium (Sigma, St. Louis,MO, USA) plus 10% fetal bovine serum (FBS) (Invitrogen, San Diego,CA, USA), respectively. Cells were cultured overnight. The mediumwas refreshed with new medium containing 0, , , , ,, , 25, 50, 100, and 200 mM FSCPs; cells wereincubated for 48 hours before MTT and procollagen type I productionassays. For MTT assay, 100 ul of the MTT solution [amixture of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and yolk lipoprotein (YLP) (Invitrogen, San Diego,CA, USA) in PBS] was added into wells and reacted for 3 hours at37,,C. The plates were stirred for 2 min, and correspondingabsorbance was recorded at 570 nm using an ELISA reader [6].For the procollagen type I production assay, the procollagen type IC-peptide ELISA kit (Takara Bio Inc, Otsu, Japan) was used.Experimental procedures followed the manufacturer,,s instruction. Inshort, 20 ul of culture medium and 100 ul ofthe antibody-POD conjugate solution were sequentially added intomicrotiter plates and reacted for 3 hours at 37º C. After 4 x5 min washing with the provided washing buffersolution (400 ul/well),100 ul of thesubstrate solution was added in. After 15 min room temperatureincubation, the stop solution (100 ul) was added in; corresponding absorbance was recordedat 450 nm using an ELISA reader.. Molecular Size Effects of FSCPsThe enzymatic hydrolysates from the fish scales werefractionationed into five different peptide pools, using theMillipore minitan system (Millipore, Bedford, MA) with fourdifferent molecular weight cutoff membranes (5, 3, 1, and kDa). A 50 ul aliquot of fish scalehydrolysate was loaded onto a Superdex peptide 10/300 GL(10 mm x 300 mm x 13 um) gel filtration column (Pharmacia, Uppsala, Sweden)and eluted with 200 mM phosphate buffer (with M NaCl, pH ) at a flow rate of  mL/min.FSCPs were calibrated against protein standards (Bio-Rad,California, USA) of cytochrome C (Mr = 12500 Da),aprotinin (Mr = 6512 Da), vitamin B12 (Mr =1355 Da), andcytidine (Mr =246 Da). Average molecular weights (MW) offractionationed FSCPs were determined to be 4500, 3500, 2000, and1300 Da, respectively; each fraction was lyophilized foruse.. FSCPs Labeled with Fluorescein IsothiocyanateFITC-labeled FSCPs were obtained by using the Fluoro Tag FITCconjugation kit (Sigma, St. Louis, MO, USA). The FITC-labeled FSCPswere prepared based on a conjugation reaction between theisothiocyanate group in FITC and the primary amino groups incollagen peptides [23]. In short, FSCPs solution (750 ppm,1 mL) was rebuffered with a phosphate buffer solution(pH ). The new FSCPs solution was mixed with  mL of M sodium carbonate-bicarbonate buffer (pH )containing FITC, which was gently stirred for 2 hours at roomtemperature. The FSCPs labeled with FITC were isolated by SephadexG-25 column (Bio-Rad, California, USA), where phosphate saline(10 mM sodium phosphate, 27 mM KCl, 138 mM NaCl,and pH ) was used as the mobile phase.. In Vivo Transdermal Delivery Efficiency of FITC-LabeledFSCPsSix-week-old female C3H/HeN mice were anesthetized usingacepromazine maleate (.). Hairs covering the areas of abdomenskin were removed with a shaver. Residual hairs were removed byhair-remove-cream (Yanagiya, Japan). Mice were subjected todestratum corneum by treating mice with 10% alpha hydroxyl acids(AHAs; BIOPEUTIC, USA) for 5 minutes and cleanup for use. Analiquot of 25 ug FITC-labeled FSCPs in sterile DI water(100 ul) was introduced into nonwoven fabrics (cosmeticsmask; Widetex Biotech Co., Taiwan). The fabric covered1 cm2 of hairless dorsal back skin which then wastopped with  cm2 transparent dressing film (Tegaderm,Neuss, Germany). The nonwoven fabric was removed after one hour.The skin tissues treated with the FITC-labeled FSCPs were embeddedin . Embedding Medium (Sakura Finetek USA, Torrance,California) and sectioned into 10 um thickness. The skintissues were observed under fluorescence microscopy (BX-51,Olympus, Japan) equipped with a digital camera [24].. The Franz-Type Diffusion Cell ModelThe procedure was modified from the method described by Kim etal. [25]. Nude mice skin was mounted on the receptor compartment ofthe Franz-type diffusion cell (PermeGear, HT, USA). A phosphatebuffer solution ( g/l potassium phosphate monobasic, g/l sodium chloride, and  g/lsodium acid; pH ) containing 750 ppm a given fraction of FSCPswas added in the tight interface, facing nude mice skin. The donorcap was filmed with a parafilm. An isotonic phosphate buffersolution (pH ) was used as the receptor solution. The receptorsolution was stirred by a magnetic follower rotating at250 rpm (which would increase mixing efficiency andreduce tendency of forming a stagnant boundary layer next tomembrane surface). Samples were performed at 1, 2, 3, 4, 6, and 24hours. The amount of total collagen peptides from the receptorsolution was measured by the BCA protein assay (Pierce, Rockford,USA).The cumulative penetration amount (ug) = [C (ppm) x Vi (mL)] +,,n-1(C x Vs)(C= concentration; Vi = initial volume; Vs= samplingvolume).The unit area cumulative penetration amount (ug/cm2) =cumulative penetration amount/the measure of area ( cm2).. Transdermal Penetration Efficiency of FITC-Labeled FSCPsDetermined by Confocal Spectral MicroscopeVarious sizes of FSCPs were labeled with FITC, which wouldpenetrate into nude mice skins in the Franz-type diffusion cellmodel. The skins of nude mice treated with FITC alone served as acontrol. After one hour, the treated skin tissues were dissectedout, embedded in . compound and sectioned in 10 umthickness. The slides were then mounted on and treated with ablocking solution (95% ethanol and 5% acetic acid). After rinsedwith PBS for 20 seconds and then rinsed with 50%, 70%, 90%, and100% ethanol for 30 seconds, the slides were observed under aconfocal spectral microscope (TCS SP5, Leica, Wetzlar,Germany).. Statistical AnalysisThe graphs and statistical analyses were performed usingSigmaPlot and SigmaStat. The statistical analyses between groupswere determined by nonparametric one-way analysis of variance(Krmskal-Wallis test) and Mann-Whitney tests. Differences wereconsidered significant if the value was ,, .05.3. Results. Effects of FSCPs on Human Facial Skin Moisture Contents andRelative ElasticityWhile collagen peptides have been well documented to be an idealmaterial in cosmetic industries [26], fish scale collagen peptides(FSCPs) as a sustainable collagen source however did not draw greatattention. In this study, we examined the treating effects of FSCPsas a major component in a skin essence on the facial skin moisturecontents and relative elasticity by using standard professionalskin probes. The FSCP-based skin essences were first formulatedinto 5%, 7%, and 10% of total FSCPs as the common basalingredients. Sixty-two voluntary Taiwanese women (within 23 to 60years of age) were subjected to the FSCP-based skin essencetreatment on facial skin twice a day for 30 days. The effects withall FSCP-based skin essences on the skin moisture contents werefound improving significantly but in a time- and dose-dependentmanner during the testing course (P,, .05; Figure 1(a)). Likewise,the effects with all FSCP-based skin essences on the relativeelasticity of facial skin were also found increasing in the samemanner as for the skin moisture test by 24%, 33%, and 35%,respectively (P,, .05; Figure 1(b)). Thereby, the FSCP-based skinessences did improve facial skin qualities (moisture andelasticity) in a time- and dose-dependent manner in these testedsubjects.fig11a1bFigure 1: Effects of FSCP-based skin essence on moisturecontents and relative elasticity of facial skins. The skin moisturecontents (a) and relative skin elasticity (b) were measured by theprofessional skin measurement probes. Bars are expressed asmeans,,SEM in each group for the 62 subjects. Difference letters inthe same group indicate significant difference (P,,.05).. Cell Proliferation and Collagen Release in Embryonic SkinFibroblast Cell Lines after FSCPs TreatmentsTo determine whether the skin quality improving was resultedfrom fibroblast cells proliferation and mass collagen synthesis indermis after the FSCPs treatment, model cells Detroit 551 (humanembryonic skin fibroblast) and STO (mouse embryonic fibroblast)were chosen for the purpose stated. The cells were grown in mediain the presence of various concentrations of FSCPs. On thecontrary, the cells grown in media without FSCPs served ascontrols. Cell proliferation and procollagen release in the testedcells were determined by MTT assays and procollagen type IC-peptide ELISA assays, respectively. As shown in Figure 2(a),FSCPs (–200 ug/mL) can dose-dependently stimulate the cellproliferation in both fibroblast cells. Likewise, the procollagentype I synthesis was found mass produced in aconcentration-dependent manner in the cells with FSCPs treatments(Figure 2(b)). The procollagen synthesis in the presence of200 ug/mL FSCPs was found most prominent and peaked ashigh as 250%. FSCPs thereby were determined to be able toeffectively stimulate and induce fibroblast cell proliferation andcollagen synthesis so as to improve the facial skin quality.fig22a2bFigure 2: Cell proliferation and collagen release in embryonicskin fibroblast cell lines treated by FSCPs. Comparisons of theproliferation activity (a) and procollagen synthesis ability (b) onDetroit 551 cells (human embryonic skin fibroblast line) and STOcells (mouse embryonic fibroblast) treated with 0, , , ,, , , 25, 50, 100, and 200 mM FSCPs. The group withno FSCPs acts as a control. After 48 hours posttreatment, thesamples were analyzed by MTT and procollagen type I C-peptide ELISAassays. Bars stand as means ,, standard deviation (triplicate foreach group).. In Vivo Determination of Transdermal Delivery EfficiencyUsing FITC-Labeled FSCPsAs mentioned above, the stratum corneum is the outmost layer ofthe epidermis, which serves as a physical barrier, for instance,preventing pathogens from invasion and also limiting desiredmacromolecules to pass by. To evaluate whether the processed FSCPsare capable of overcoming the stratum corneum barrier, an animalmodel experiment was set to determine the transdermal efficiency ofthe prepared FSCPs. Six-week-old female C3H/HeN mice were firstshaved and treated with hair-removal cream to remove residualhairs. The mice were then subjected to treatments with or withoutalpha hydroxyl acids (AHAs, for weakening the stratum corneum)before topical application with the nonwoven fabric that contained25 ug FITC-labeled FSCPs. The mice pretreated with 10%AHAs and topped with the nonwoven fabric containing no FITC-labeledFSCPs served as controls. These treated skins were dissected outand immediately embedded with . in due course. Then, thesamples were sectioned, mounted on glass slides, and observed underfluorescence microscopy. As shown in Figure 3, the FITC positivesignals laid mainly on the area of superficial epidermis. And, theFITC positive signals in the skin pre-treated with AHA were founddeeper and brighter than those without AHA treatment. The FITCpositive signals were also found in the hair follicles ofepidermis. Thus, the stratum corneum barrier was determined to be akey factor that influences the penetration efficiency of FSCPs inmice skin. Nonetheless, FSCPs without AHA treatment stillconsiderably pass by the stratum corneum, which agreed with theresults in the human facial skin test.fig33a,3b,3c3d,3e,3f3g,3h,3iFigure 3: Fluorescence microscopic images of FITC-labeled FSCPstreated C3H/HeN mice skins. The green fluorescence is found in thefollicular and epidermal areas of the vertical cross-sections ofthe treated skins. (a), (d), and (g) are the phase contrasts of thetreated skins; (d)–(f) are groups with FITC-labeled FSCPs butwithout AHA pretreatment; (g)–(i) are groups with FITC-labeledFSCPs and with AHA pretreatment. (40x, 40-fold magnification; 100x,100-fold magnification).. Transdermal Penetration Ability ofFSCPs in Franz-Type Diffusion Cell ModelWe considered factors suchas molecular volume (MV) or molecular weight (MW) of collagens mayplay a role on their penetration capability when administratedtopically. The size effect for given FSCPs was determined byemploying the Franz-type diffusion cell model. Fish scalehydrolates were first subjected to gel chromatography (superdexpeptide 10/300 GL columns), whereby FSCP hydrolysates werefractionationed by sizes. As shown in Figure 4(a), the elutedpeptides were collected into six fractions, > KDa(%), 3– KDa (%), – KDa (%), – KDa(%). – KDa (%) and < KDa (%).These peptide fractions were further subjected to ultrafiltrationmembrane refining (5, 3, 1, and  kDa cutoff) into four refinedgroups I–IV (Mw = 3–5, 1–3, 1–, and< KDa) with average molecular weights of4500, 3500, 2000, and 1300 Da, respectively (Figure4(b)). These collagen peptides were corroborated by gelchromatography (Sp-Sephadex C-25 column) against standard markers.As shown in Figure 5, all groups were found to be able to penetratethe nude mice skin in the Franz-type diffusion cell model but invarious extents and in a time-dependent manner. In short, groups Iand II exhibited relatively better transdermal penetration abilityin the first 4 hours. The highest cumulative penetration amountsfor groups I and II at 24 hours were ,, and ,, ug/cm2, respectively. In contrast, theFSCPs mixture (total FSCP hydrolates without ultrafiltrationmembrane refining) showed the lowest transdermal penetrationcapability (1010 ,,  ug/cm2) (Figure 5).fig44a4bFigure 4: Size distribution of FSCPs. (a) Fish scalehydrolysates were separated by gel chromatography (superdex peptide10/300 GL columns); molecular sizes were determined for eachfraction; size distributions were plotted accordingly. The sizedistribution (KDa) is expressed as means ,, SEM. (b) Size profilesof FSCPs. Protein standards (Bio-Rad, California, USA) arecytochrome C (Mr =12500 Da), aprotinin (Mr = 6512 Da),vitamin B12 (Mr =1355 Da), and cytidine (Mr =246 Da). The average molecular weights (MWs) weredetermined to be 4500, 3500, 2000, and 1300 Da forthe four indicated groups.Figure 5: Transdermal permeation abilities of given FSCPsfractions. The permeation abilities of various molecular sizes ofFSCPs were determined by the Franz-type diffusion cell model,performing at 1, 2, 3, 4, 6, and 24 hours. The amounts of totalcollagen peptides from the receptor solutions were determined bythe BCA protein assay. ,,Mixture,, indicates the FSCP hydrolateswithout ultracentrifugation membrane treatment. The results shownare a representative in 3 experiments.. Transdermal Penetration Depth and Peptide Conformation inNude Mice SkinTo probe the penetration depth and peptide conformation forgiven FSCPs, each FSCPs group was first labeled with FITC and thenapplied to nude mice skins in the Franz-type diffusion cell modelfor one hour. The skin treated with given FITC-labeled FSCPsproceeded according to the same procedure described in the previoussection. Interestingly, groups I (Figure 6(d)) and II (Figure 6(c))exhibited a coil-like structure in epidermis as opposed to groupsIII (Figure 6(b)) and IV (Figure 6(a)) which showed less folded orin a linear form. In contrast, the mixed FSCPs did not have adefined structure (Figure 6(e)). The penetration depths for eachFSCPs group were also measured by confocal spectral microscopy. Thedistances for groups IV–I and the mixture were , , ,, and  um, respectively. The heavier FSCPs penetrateddeeper than the lighter. As a result, the transdermal penetrationefficiency of given FSCPs are positively correlated to FSCPs,, sizesand/or conformations thereof. The folded structure seemed to helppenetration, which however is an interesting issue for futurestudy.fig66a, 6b, 6c6d, 6e, 6fFigure 6: Peptide conformations of various sizes of FITC-labeledFSCPs in nude mice skins. Nude mice skins were treated with variousmolecular sizes of FITC-labeled FSCPs in the Franz-type diffusioncell model. The topical sites of tested nude mice skins werecollected in one hour. The vertical cross-sections of the nude miceskins were imaged for FITC signals using cryosection and confocalspectral microscope systems. (a) 1,300 Da FSCPs; (b)2,000 Da FSCPs; (c) 3,500 Da FSCPs; (d)4,500 Da FSCPs; and (e) the mixture FSCPs (FSCPs withoutultracentrifugation membrane treatment).4. DiscussionMany reports have pointed out collagen,,s versatile applications,prominently for cosmetic industry [13]. Fish scale collagens appearto be an ideal collagen source as they are cheap, rich andsustainable [25, 26]. Since collagen is the key component inmaintaining the elasticity of skin, diminishing collagen contentsin skin would therefore result in wrinkled and flabby skins [27].In this paper, we have demonstrated the formulated FCSP-based skinessence possesses such desirable effects as increasing skinmoisture contents and relative elasticity (Figure 1). The FCSPs at200 ug/mL provided the best effects by manifestingembryonic skin fibroblast cell proliferations (Detroit 551 cellsand STO cells) and procollagen synthesis. These results otherwiseare reinforced by the reports of Katayama et al., wherein theydemonstrated pentapeptides from type I collagen promotedextracellular matrix production in fibroblasts [28, 29].Since the stratum corneum acts as a physical barrier formultiple purposes in skins [19, 20], it does influence thepenetration efficiency of the FSCP-based skin essences as they werepresent mainly in the surface of epidermis. However, givenFITC-labeled FSCPs still effectively passed by stratum corneum toepidermis and dermis in the mice model experiment. The reports ofPotts and Guy agreed with the results, in which they proposed thediffusivity of a drug in skin is correlated to given molecularfeatures of the drug [30, 31], although Lin et al. [32] consideredthe effect of lipophilicity in the passive diffusion permeabilityis the dominant factor other than the compound,,s molecular weight.In addition, Potts and Guy further proposed that the higher themolecular weights the greater the lipophilicity/permeability [30].The better effects of the FSCPs,, groups at Mw 3500 and Mw4500 Da may possess desired sizes and effectivestructural features. Furthermore, collagens typically exist as aright-handed triple helix structure of Gly-X-Y helix-formingrepeats in a length of 300 nm which corresponds to 1000amino acids or so in fibril-forming collagens (I, II, III) [33–36].Fish scale collagens are within the scope of the type I collagen[26]. The higher penetration efficiency of FSCPs at 3500 and4500 Da may thus be attributed to the synergisticeffects of high lipophilicity (adequate size) and constructivestructure features.In conclusion, the prepared FSCPs haveshown to be able to effectively penetrate stratum corneum toepidermis and dermis. FSCPs at epidermis and dermis in situ canactivate fibroblasts and accelerate collagen synthesis, whereby thefacial skin qualities (moisture contents and relative elasticity)were significantly improved. As a consequence, the prepared FSCPsalong with the formulated skin essence should hold high commercialinterests and are worthy of further in-depth study for enhancingFSCPs transdermal penetration ability to a higher level.References1. T. Nagai, Y. Araki, and N. Suzuki, ,,Collagen of the skin ofocellate puffer fish (Takifugu rubripes),,, Food Chemistry, vol. 78,no. 2, pp. 173–177, 2002. View at Publisher ,, View at GoogleScholar ,, View at Scopus2. R. Mayne and R. G. 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