Antioxidant Graphene Oxide Nanoribbon Raws li Novel Whitening Agent Inhibits Melanogenesis Mechanism

Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Hsin-Yu Chou, Hui-Min David Wang,* Chia-Heng Kuo, Pei-Hsuan Lu, Lin Wang, Wenyi Kang, thiab Chia-Liang Sun*

TSEEM CEEB:Hauv cov txheej txheem melanin synthesis, cov tshuaj tiv thaiv oxidative ua lub luag haujlwm tseem ceeb, thiab nws yog lub tswv yim zoo los tiv thaiv melanin ntau lawm los ntawm kev txo oxidative kev nyuaj siab. Fullerene thiab nws cov derivatives, los yog cov complexes, raug suav hais tias yog muaj zog dawb-radical scavengers, thiab peb ntxiv siv multilayered sp2 nanocarbons mus nrhiav tau.melaninsynthesis inhibitory mechanisms. Nyob rau hauv txoj kev tshawb no, peb siv cov nanomaterials tshiab, xws li multiwalled carbon nanotubes (MWCNTs), luv-hom MWCNTs, graphene oxide nanoribbons (GONRs), thiab luv-hom GONRs, ua cov tshuaj tiv thaiv oxidative los tswj melanin ntau lawm. Cov txiaj ntsig tau pom tias GONRs muaj peev xwm tiv thaiv oxidative zoo dua nyob rau hauv intracellular thiab extracellular oxidative stress analysis platforms dua lwm tus. Peb tau thov tias GONRs muaj cov pab pawg ua haujlwm oxygen. Hauv 2′,7′-dichlorodihydrofluorescein diacetate assay, peb pom tias GONR tuaj yeem chelate hlau ions kom tshem tawm cov pa oxygen reactive. Hauv kev pom kev pom ntawm molecular, peb tau pom tias cov nanomaterials downregulated melanin synthesis los ntawm kev txo qis microphthalmia-txog kev hloov pauv yam cuam tshuam txog cov noob caj noob ces, thiab muaj cov txiaj ntsig zoo sib xws hauv cov lus qhia protein. Txhawm rau ua kom tiav, GONRs yog tus neeg sawv cev muaj peev xwm ua cov tshuaj tua kab mob tshiab thiab cov khoom siv tshuaj pleev ib ce.

Cistanche kuj yogtus neeg sawv cev muaj peev xwm ua tus tshiabantioxidantthiab tawv nqaij dawbkhoom siv cosmetology.

1. Kev Taw Qhia

Daim tawv nqaij yog lub cev uas npog sab nrauv ntawm tib neeg lub cev. Txij li thaum lub interface yog nyob rau hauv kev sib cuag nrog ib puag ncig, cov tawv nqaij txheej plays lub luag haujlwm tseem ceeb hauv kev tiv thaiv lub cev tiv thaiv kab mob, tsis txhob poob dej ntau, tswj lub cev kub, thiab lwm yam. Melanocytes loj hlob hauv daim nyias nyias ntawm daim tawv nqaij epidermis thiab suav txog 5 feem pua ​​​​rau 10 feem pua ​​​​ntawm cov ntsiab lus ntawm tes. Lawv tau raug tus cwj pwm li unicellular "cov qog" muaj nyias, ntev, streamer-zoo li dendrites thiab branching. Melanocytes txav mus los ntawm cov hlwb epidermal hauv lawv thaj tsam tam sim ntawd, tsim ib lub hnub qub ntawm cov hlwb epidermal nyob ib ncig ntawm txhua lub melanocyte. Muaj ntau qhov ua rau sab hauv thiab sab nraud ntawm daim tawv nqaij laus, thiab ib qho zoo li no yog ultraviolet (UV) hluav taws xob los ntawm lub hnub ci.1 Thaum lub sij hawm UV raug, cov qib reactive oxygen (ROS) hauv daim tawv nqaij nce ntau, uas yog hu ua oxidative stress. Ob peb yam toxicity ib puag ncig kuj txhim kho oxidative kev nyuaj siab rau ntawm daim tawv nqaij, xws li tshuaj tua kab, carbon tetrachloride, hnyav hlau, aromatic amines, thiab particle teeb meem 2.5 (PM2.5). enzymatic system, hloov lawv mus rau hauv ROS ua rau lub melanogenesis txoj kev.3

Ntxiv rau ROS, muaj ntau yam cuam tshuammelanin ntau lawm, suav nrog cov noob qhia, mob, hloov pauv endocrine, thiab pigment uptake.1 Hauv thawj kauj ruam ntawm kev tsim melanin, tyrosinase plays lub luag haujlwm hauv catalyzing tyrosine rau hauv phenomelanin thiab eumelanin. Ob lub pigments manufacturing mechanisms zoo sib xws, uas suav nrog L- tyrosine hydroxylation rau 3, 4-dihydroxy-L-phenylalanine (L- DOPA) thiab L-DOPA oxidation rau dopaquinone. Hauv cov kauj ruam tom ntej, dopamine yog oxidized los ntawm tyrosinase-related protein 1 (TRP-1) thiab tyrosinase-related protein 2 (TRP-2) nyob rau hauv melanosome, uas yog tswj los ntawm microphthalmia-uas cuam tshuam kev hloov pauv ( MITF) los tsim melanin. Thaum kawg, melanin yog matured thiab precipitated nyob rau hauv lub stratum corneum.4,5 Cov no nkag mus rau cov nyob sib ze keratinocytes ntawm basal txheej thiab tiv thaiv lawv DNA los ntawm tej UV-induced mutations los yog hloov. Lub matured melanin nyob rau hauv melanosomes yog pauv mus rau keratinocytes6-9 thiab thaum kawg ua rau lub pigmentation ntev. Lentigines, freckles, thiab xim av / dub me ntsis ua rau muaj teeb meem kev sib raug zoo ntawm cov txiv neej thiab poj niam qee zaum. Thaiv cov oxidative kev nyuaj siab los yog suppressing tyrosinase kev ua si yog ib lub tswv yim rau downregulate lub syndrome ntawm hyper-pigmentation thiab dermatological mob. Antioxidants kho ROS-ua rau hyper-pigmentations thiab cellular puas.10,11 Yog li ntawd, synthesized anti-oxidative tebchaw muaj ntau biofunctional daim ntaub ntawv nyob rau hauv daim ntawv thov ntawm daim tawv nqaij.

Fullerene (C60), carbon nanotube (CNT), graphene, thiab graphene nanoribbon (GNR) yog plaub hom sp2 nanocarbon dav tshawb fawb thoob plaws ntiaj teb.12 Fullerene thiab nws cov derivatives lossis complexes tau pom tias muaj zog.dawb radical scavengersntev. Yod et al. siv dej-soluble C60 ua tus neeg sawv cev tiv thaiv catabolic stress-induced degeneration. Txhaj thiab al. xaus lus tias C60 (OH) 24 yog ib qho muaj zog antioxidant compound thaum lub zog oxidative siab dhau. Okuda et al. qhia tias C60 complexes tuaj yeem tiv thaiv NO-mediated cell raug mob.13,14 Tong et al. qhia tau tias C60complexes yuav tau cog lus rau cov neeg sib tw los kho cov kab mob hauv hlwb los ntawm kev nce qib ntawm superoxide. Qhov tseeb, ib lub tuam txhab Nyij Pooj tau txheeb xyuas cov fullerenes nrog lub zog antioxidant kev ua kom zoo nkauj hauv xyoo 2006. Lucente- Schultz li al. pom tau tias cov pa oxygen radical scavenging peev xwm ntawm functionalized ib leeg-walled CNTs (SWCNTs) yog ze li ntawm 40 npaug ntau dua li dendritic C60.15-19 Fenoglio li al. Pom tias multiwalled CNTs (mwcnts) muaj ib tug zoo tshaj plaws radical muaj peev xwm sib cuag tau muaj cov qauv ntawm swcnts raws li dawb-radical scavengers. Nyob rau hauv 2004, Novoselov et al. thawj zaug pom tau tias graphene tau nthuav tawm lub zog ambipolar hluav taws xob thiab tuaj yeem cog lus rau kev siv hluav taws xob.21 Tom qab ntawd, lawv txuas ntxiv qhia tias graphene muaj cov khoom siv hluav taws xob uas txawv ntawm 2D roj ntawm cov lus piav qhia los ntawm Dirac equation.22,23 Txij li thaum ob cov ntaub ntawv tseem ceeb no, ntau thiab ntau dua tau muab rau kev tshawb fawb graphene.24-30 Piv txwv li, Qiu li al. Hauv xyoo 2014 tau pom tias graphene oxide thiab ob peb txheej graphene nthuav tawm cov haujlwm tseem ceeb antioxidant thiab tuaj yeem tiv thaiv ntau yam biomolecular molecules los ntawm oxidation.31 Han li al. sim ua kom pom nyob rau xyoo 2007 tias qhov sib txawv ntawm lub zog ntawm GNRs tuaj yeem tswj tau thaum lub sij hawm lithography txheej txheem los ntawm kev hloov cov ribbon dav.32 Ntawm plaub nanocarbons, GNRs tau txais qhov tsawg tshaj plaws. Rau peb txoj kev paub, muaj kev tshawb fawb me me ntawm cov khoom antioxidant ntawm graphene oxide nanoribbons (GONRs).31,33 Li no, hauv txoj kev tshawb no, peb ua tib zoo npaj MWCNTs, luv MWCNTs, GONRs, thiab luv GONRs thiab tsom los sib piv lawv cov khoom antioxidant thiab cuam ​​tshuam cov txiaj ntsig systematically.

2. TAM SIM NO THIAB PAUB TIAS

2.1. Morphology ntawm MWCNTs thiab GONRs.

Figure 1a shows the low- and high-magnification transmission electron microscopy (TEM) images of MWCNTs and short MWCNTs. Following acidic cutting under ultrasonication, the length of MWCNTs could be shortened from >1 0 μm rau 2-3 μm. Nyob rau tib lub sijhawm, nws tau pom tias kev kho nitric acid roughens lub raj du nto. Qee qhov tsis sib xws thiab cov duab tsis sib xws tau tshwm sim hauv daim duab loj loj. Tsis tas li ntawd, siv MWCNTs thiab luv MWCNTs los ntawm microwave cov tshuaj tiv thaiv tau txais GONR thiab luv GONR, feem. Peb kuj tau piav qhia qhov qis- thiab siab-loj TEM duab ntawm GONR thiab luv GONR. Raws li qhov loj longitudinal unzipping thiab me kab rov tav txiav, nws zoo li GONRs luv dua MWCNTs. Ntawm qhov tod tes, cov duab loj loj tau pom cov kab uas loj dua, uas yog, 0.11-0.18 μm, ntawm GONRs dua li cov MWCNTs, qhia tias cov txheej txheem unzipping tau ua tiav. Ib yam li ntawd, luv GONRs nthuav tawm qhov ntev thiab qhov loj dua li luv MWCNTs. Nyob rau hauv lub tshuab cua compressor ntawm peb cov txheej txheem unzipping tshiab, cov txheej txheej nyias nyias ntawm GONRs tsawg dua qhov peb tau txais hauv tsab ntawv ceeb toom thaum ntxov rau tib lub zog microwave ntawm 250 W thaum tuav cov tuab tuab hauv nruab nrab MWCNTs.12 Qhov no txhais tau tias yog ib qho tseem ceeb-plhaub MWCNT. /GONR heterostructure yuav tshwm sim ntau dua es tsis txhob tag nrho unzipped nanoribbon qauv los ntawm tag nrho cov microwave powers nyob rau hauv tus txheej txheem tshiab. Txhawm rau sib piv nrog GONR luv luv hauv peb cov kev tshawb fawb yav dhau los, 34 lub zog microwave siab dua tau tsim ntau qhov chaw nyob ntawm sab ntawm ribbons thiab tsis tsim cov npoo zoo, du ribbon. Nco ntsoov tias peb siv ob yam sib txawv ntawm Cu daim phiaj hauv daim duab 1a. Rau MWCNTs thiab GONRs nrog ntev txaus, Gu grid nrog lacey formvar stabilized nrog carbon (khoom no. 01881-F, Ted Pella, Inc., USA) tau siv. Qhov qhib qhov nyob rau hauv cov zaj duab xis lacey carbon tiv thaiv cov duab sib tshooj ntawm nanocarbons thiab cov yeeb yaj kiab carbon. Cov tes hauj lwm grey tsaus yog nyob rau hauv cov yeeb yaj kiab lacey. Txawm li cas los xij, Gu grid nrog formvar stabilized nrog carbon (cov khoom no. 01800-F, Ted Pella, Inc., USA) yog xav tau rau luv luv MWCNT thiab luv GONR. Qhov no yog vim qhov loj hauv cov yeeb yaj kiab lacey carbon ua rau muaj teeb meem rau tuav lub luv luv MWCNT thiab GONR luv. Raws li pom nyob rau hauv daim duab 1, lub teeb grey sib piv nyob rau hauv lub luv luv MWCNTs thiab luv GONRs yog lub teeb txheej ntawm carbon. Cov pa roj carbon txheej no ruaj khov rau cov yeeb yaj kiab formvar raug rau cov kab hluav taws xob ntawm nws cov khoom siv hluav taws xob thiab hluav taws xob.

2.2. Bonding Configurations ntawm MWCNTs thiab GONRs.

Raman spectra ntawm plaub nanocarbons tau nthuav tawm hauv daim duab 1b; D band ntawm GONRs siab dua li ntawm MWCNTs tom qab cov txheej txheem unzipping. Qhov no tau raug ntaus nqi rau qib oxidation ntau dua thiab ntau dua ntawm ntug qauv ntawm GONR piv rau MWCNTs. Qhov tshwm sim no kuj zoo ib yam li qhov peb tau pom hauv 2011.12 Raws li qib graphitization siab, G band ntawm MWCNTs muaj qhov qis tshaj plaws-dav-ntawm- ib nrab-ntau tus lej. Tus ID/IG piv ntawm plaub nanocarbons yog {{10}}}076, 0.502, 0.483, thiab 0.700, raws li. Luv luv, qhov ntev ntawm qhov txo qis thiab oxidation saum npoo tau nce qhov tsis xws luag thiab yog li ua rau ID / IG ratios siab dua. D' ncov yog tam sim no nyob rau hauv tag nrho cov graphenes tsis zoo thiab yog saib raws li ib tug kev ntsuas ntawm zoo.35 Raws li qhia nyob rau hauv daim duab 1b, lub D' ncov nyob rau hauv lub plaub spectra ua ib tug tseem ceeb tom qab txiav los yog unzipping txheej txheem, qhia tias lawv puas. cov txheej txheem uas qhia ntau yam tsis xws luag. Daim duab 1c,d tso saib X-ray photoelectron spectroscopy spectra ntawm plaub nanocarbons. Thaj, D' ncov yog qhov tseeb tshaj plaws rau luv GONRs. Raws li pom nyob rau hauv daim duab 1c, O qib tau nce los ntawm 7.6 feem pua ​​(MWCNTs) mus rau 19.9 feem pua ​​(GONRs) vim muaj zog oxidation muaj peev xwm ntawm KMnO4 nyob rau hauv ib puag ncig acidic. Ntawm qhov tod tes, O qib me ntsis nce los ntawm 0.8 feem pua ​​​​ntawm MWCNT mus rau luv luv MWCNTs. Qhov tseem ceeb, qib O siab tshaj plaws yog 38.3 feem pua ​​​​rau luv luv GONR, txhais tau hais tias qhov kawg ntawm cov nanoribbons yuav yooj yim dua los txuas cov pa oxygen ua haujlwm ntau dua li cov txheej txheem sp2. Qhov loj dua qhov dav-ntawm-ib nrab-ntau tshaj tus lej thiab kev hloov mus rau lub zog siab khi ntawm C 1s peaks tom qab cov txheej txheem unzipping ntawm ob qho tib si MWCNTs thiab luv MWCNTs yog qhia hauv daim duab 1d. Rau graphene oxides, lub deconvoluted peaks nyob rau hauv high-binding-zog sab yuav muab rau C-C (CC), C-O, CO, thiab COOH bondings.36 Peb characterized GONR (200 W) nyob rau hauv 2013 ,37 thiab cov txiaj ntsig tau zoo ib yam li qhov kev tshawb fawb no. Txoj kev tshawb no xaus qhov tshwm sim ntawm Raman spectra, txhais tau hais tias ntau cov pa oxygen-muaj cov haujlwm ua haujlwm tau tsim tawm thaum lub raj-rau-ribbon hloov pauv (Daim duab 2).

2.3. Anti-oxidative Properties ntawm MWCNTs thiab GONRs.

2.3.1. Kev txiav txim siab ntawm 1,1-Diphenyl-2-picrylhy- drazyl Dawb Radical Scavenging Activity Assays.

1,1- Diphenyl-2-picrylhydrazyl (DPPH) dawb-radical scavenging kev ua si yog ib qho antioxidant platform siv los kuaj xyuas lub peev xwm tiv thaiv oxidative; Cov txiaj ntsig ntawm plaub nanocarbons tau piav qhia hauv Table 2. Hauv DPPH kev soj ntsuam, vitamin C ntawm qhov concentration ntawm 100 μM tau siv los ua kev tswj hwm zoo. Txhawm rau kuaj cov haujlwm tiv thaiv oxidative ntawm MWCNTs, luv MWCNTs, GONRs, thiab luv GONRs, ntau npaum li cas ntawm 1, 5, thiab 10 mg / L tau incubated rau hauv cov tshuaj tiv thaiv los ntsuas cov khoom. MWCNTs, luv MWCNTs, GONRs, thiab luv GONRs muaj peev xwm inhibitory nruab nrab ntawm 10 mg/L (19.2 ± 0.3, 12.1 ± 0.3, 26.8 ± 0.3, thiab 30.0 ± 0.4 feem pua), thaum cov vitamin C muaj qhov zoo sib xws ntawm 100 μM (93.4 ± 0.1 feem pua) rau kev tawm tsam.

2.3.2. Ion-Chelating Activity Assay.

Hauv qhov xwm txheej oxidative kev ntxhov siab, ferrozine tuaj yeem tsim ib qho nyuaj nrog Fe2 ntxiv rau kev ntsuas ntau. Nyob rau hauv lub xub ntiag ntawm chelating mediators, lub complex yog tawg, ua rau cov ferrous ions txo los ntawm ib tug tsaus liab xim ntawm lub Fe2 ntxiv complex. Peb siv EDTA ua tus tswj zoo. Table 2 qhia tau hais tias MWCNTs, luv MWCNTs, GONRs, thiab luv GONRs muaj chelating kev ua ntawm 10 mg/L (29.2 ± {{10}}.8, 28.7 ± 0 .7, 69.7 ± 0.6, thiab 68.9 ± 0.3 feem pua ​​​​), thaum qhov kev tswj zoo muaj qhov zoo sib xws ntawm 100 μM (93.4 ± 0.1 feem pua).

2.3.3. Ferric-txo Antioxidant zog ntsuas.

Qhov kev ntsuam xyuas ferric-txo muaj peev xwm yog qhov kev sim yooj yim thiab txhim khu kev qha siv los ntsuas qhov Fe (III)-ferricyanide complex synthesis. Hauv qhov kev ntsuam xyuas no, qhov txo qis zog ntawm plaub nanocarbons uas tsim cov ferrous Fe (III)-TPTZ complex tau kuaj pom los ntawm kev hloov xim ntawm cov tshuaj los ntawm daj mus rau ntsuab thiab xiav. Table 2 qhia tau hais tias qhov txo hwj chim ntawm MWCNT, luv MWCNT, GONR, thiab luv GONR yog qhov muag pom kev ceev (OD) 1.11, 1.13, 1.15, thiab 1.11 ntawm 10 mg / L.

2.3.4. MWCNTs thiab GONRs Inhibit Intracellular ROS Accumulation.

Ntau cov ntawv ceeb toom tau pom tias ROS rhuav tshem cov txheej txheem kev ncaj ncees ntawm cov cell membranes, suav nrog cov cell membranes thiab nuclear membranes ua rau cov cell puas thiab tsis ua haujlwm li qub.38-40 Tsis tas li ntawd, ROS kuj yog ib qho tseem ceeb rau catalyze tyrosinase tsim. melanin, thiab inhibition ntawm ROS ntau lawm yog lub tswv yim zoo rau downregulate melanin synthesis. Nyob rau hauv txoj kev tshawb no, peb siv 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining assay los soj ntsuam cov intracellular oxidative kev nyuaj siab nyob rau hauv MWCNT thiab GONR kho hlwb. Phorbol 12-myristate 13-acetate (PMA) induced oxidative stimulations nyob rau hauv MWCNT thiab GONR pawg thiab tau siv raws li kev tswj tsis zoo.41 Thaum lub concentration ntawm PMA yog 20 ng / mL, nws induced oxidative kev nyuaj siab, nce. tus nqi rau 38 feem pua; Tom qab kho GONRs thiab MWCNTs, theem ntawm ROS raug txo qis mus rau qib qub. Cov ntaub ntawv qhia tau hais tias ob qho tib si cov ntaub ntawv inhibit oxidative kev nyuaj siab, thiab cov nyhuv anti-oxidative ntawm GONRs yog siab dua li ntawm MWCNTs (Daim duab 3). Table 1 qhia cov npe zoo sib xws. Peb tau sib cav tias muaj peb qhov laj thawj rau peb qhov kev tshawb pom tshiab: ua ntej, qhov kev txiav txim ntawm solubility ntawm cov ntaub ntawv no yog raws li nram no: luv GONRs > GONRs > luv MWCNTs > MWCNTs, lub ntsiab lus hais tias qhov chaw sib cuag ntawm luv GONRs yog qhov loj tshaj plaws, yog li nws yog superior rau ROS scavenging. Thib ob, GONRs thiab MWCNTs yog sp2- cov qauv carbon uas tuaj yeem rhuav tshem ROS hluav taws xob los ntawm kev tsim cov khoom siv hluav taws xob lossis hloov hluav taws xob.42 Peb pom tias cov tshuaj tiv thaiv antioxidant ntawm cov qauv nanoribbon zoo dua li cov qauv nanotube, yog li nanoribbons ua rau nws. yooj yim rau hloov electrons tshaj nanotubes. Thaum kawg, hauv daim duab 1b, peb pom tias GONR sp2-carbon site muaj cov pa oxygen ua haujlwm ntau dua li MWCNTs, pawg carboxylic acid tuaj yeem chelate hlau ions, thiab pawg hydroxyl tuaj yeem yog H-pab pab txhawm rau txhawm rau ROS thiab inhibit melanin ntau lawm.

2.4. Cytotoxicity ntawm MWCNTs thiab GONRs kho nyob rau hauv tib neeg Dermal Fibroblast Cells.

Txoj kev {{0}}}(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) txoj kev tau siv los ntsuas qhov cytotoxic thaj chaw ntawm GONRs ntawm Hs68 hlwb (Daim duab 3), thiab cov hlwb raug coj los ntawm ntau koob tshuaj ntawm 1, 5, thiab 10 ug / mL. Peb tau soj ntsuam tias cov cell viabilities ntawm MWCNT yog 100.7 ± 3.7, 99.8 ± 4.9, thiab 94.1 ± 4.7 feem pua ​​ntawm cov concentrations ntawm 1, 5, thiab 10 mg / L, raws li; viabilities rau luv luv MWCNT raug xam nyob rau hauv tib qhov kev txiav txim thiab tau pom tias yog 93.9 ± 2.2, 86.4 ± 3.0, thiab 98.9 ± 2.1 feem pua. Peb tau pom tias B16-F10 hlwb tau tsim nyob rau hauv siab concentrations, thiab cell ciaj sia taus ntawm Hs68 hlwb yog ntau tshaj 80 feem pua, qhia tias MWCNT thiab luv MWCNT tsis muaj tshuaj lom rau tib neeg dermal fibroblasts hlwb. Lub cell viabilities ntawm GONR thiab luv GONR yog 86.24 ± 2.1, 90.87 ± 3.5, 88.58 ± 2.5, 89.03 ± 3.6, 90.71 ± 2.8, thiab 90.64 ± 2.5 feem pua. Nws tseem tau taw qhia hauv daim duab 4a tias GONR thiab luv GONR tsis muaj qhov pom tau cytotoxic cuam tshuam rau HS68 hlwb. Nyob rau hauv cov ntaub ntawv dhau los, kev siv cov khoom siv tsis tau tshuaj nano rau lub hom phiaj kho kom zoo nkauj tuaj yeem suav hais tias muaj lus nug, 43,44 thiab nws feem ntau yog vim kev tawm tsam ntawm DNA tom qab cov nanoparticles nkag mus rau hauv lub hlwb. Tom qab kuaj cytotoxicity, peb pom tias peb cov ntaub ntawv tsis ua rau toxicity rau cov tawv nqaij li qub. Peb xaus lus tias tom qab nanomaterials nkag mus rau hauv lub hlwb, cov nanomaterials tsuas yog inhibit melanin ntau lawm los ntawm kev txo cov oxidative kev nyuaj siab thiab chelating hlau ions thiab tsis ua rau cov mitochondria los yog DNA, uas txhais tau hais tias MWCNTs thiab GONRs muaj kev nyab xeeb siv.

2.5. Ob Hom MWCNTs thiab GONRs hauv B16-F10 Cellular Tyrosinase Kev Ua Haujlwm thiab Melanin Cov ntsiab lus.

Nyob rau hauv txoj kev melanin synthesis, tyrosinase plays lub luag haujlwm tseem ceeb. Tyrosinase oxidizes thiab tsim eumelanin thiab pheomelanin los ntawm cov tshuaj biochemical. Txhawm rau txiav txim siab seb GONRs thiab MWCNTs cuam tshuam cov kev ua ub no ntawm tyrosinase thiab ua rau txo qis melanin ntau lawm, peb tau txheeb xyuas cov haujlwm tyrosinase hauv B16-F10 hlwb. Peb pom tias MWCNTs thiab luv MWCNTs inhibited tyrosinase kev ua haujlwm los ntawm kwv yees li 17.1 feem pua ​​thiab 23 feem pua ​​​​ntawm 10 mg / L. GONRs thiab GONRs luv luv tau muaj txiaj ntsig zoo dua hauv kev tawm tsam tyrosinase kev ua haujlwm ntawm tib qhov sib xyaw piv rau lwm GONR. Lawv kuj tau nyob rau hauv koob tshuaj thiab inhibited 49.8 feem pua ​​thiab 44.7 feem pua ​​ntawm cov tyrosinase kev ua, raws li qhia nyob rau hauv daim duab 4b.

Melanin yog ib qho tseem ceeb ntawm cov xim hauv tib neeg lub cev, tab sis qhov overexpression ntawm melanin feem ntau ua rau muaj kab mob. Hauv kev tshawb fawb yav dhau los, Xiao et al. siv cov khoom zoo sib xws, Radical Sponge, fullerene nanoparticle, ua cov tshuaj tiv thaiv melanin.45 Muaj qee qhov txiaj ntsig zoo; kwv yees li 20 feem pua ​​​​ntawm melanin ntau lawm tuaj yeem cuam tshuam. Txhawm rau txhim kho nws cov kev ua tau zoo, peb tau txhim kho cov khoom siv ntxiv los ntsuas qhov inhibition ntawm melanin thiab nws cov txheej txheem molecular, raws li qhia hauv daim duab 4c thiab 5. MWCNTs thiab luv MWCNTs txo cov ntsiab lus melanin los ntawm 17.6 ± 5.5 thiab 13.2 ± {{ 16}}.2 feem pua ​​​​ntawm 10 mg / L thiab nyob ntawm qhov koob tshuaj. GONRs thiab luv GONRs muaj zog txo qis qhov tseem ceeb rau 32.0 ± 2.3 thiab 35.3 ± 3.4 feem pua ​​​​ntawm 10 mg / L. Cov txiaj ntsig kev sim tau pom tias tag nrho plaub hom tuaj yeem cuam tshuam cov synthesis ntawm melanin thiab GONRs muaj zog dua. Ntawm qhov tod tes, peb kuj tau pom tias luv GONR muaj txiaj ntsig zoo hauv inhibiting melanin ntau lawm. Peb xaus lus tias GONRs luv luv muaj ntau pab pawg ua haujlwm thiab tuaj yeem tiv thaiv cov hlau ions-catalyzed tyrosinase, ntxiv inhibiting zus tau tej cov melanin (Daim duab 2). Hauv Table 1, peb pom tias kev siv zog ntawm cov hlau ion-chelating luv hom yog siab dua li hom ib txwm muaj; qhov no txhais tau hais tias cov GONRs luv luv no tuaj yeem siv rau hauv kev ua kom zoo nkauj ua cov neeg saib xyuas tawv nqaij.

2.6. Mechanism ntawm MWCNTs thiab GONRs Inhibited B16-F10 Cellular Melanin Cov ntsiab lus.

Cells teb rau sab nraud oxidative kev nyuaj siab los ntawm kev tswj cov protein qhia. B16-F10 hlwb txhim kho c-myc noob qhia thiab nce- tswj AMPK kom txo qis oxidative theem, 46 thiab hauv txoj haujlwm no, MITF yog ib qho kev hloov pauv tshwj xeeb ntawm tyrosinase los tswj cov molecular melanin synthesis teeb liab txoj hauv kev.47-49 Hauv daim duab 5a, MWCNTs thiab GONRs txo qis cov microphthalmia-txoj kev hloov pauv hloov pauv los ntawm kev txo cov oxidative kev nyuaj siab, thiab tom qab ntawd, cov noob caj noob ces TRP-1 thiab TRP-2 kuj tau txo qis. Rau qib protein, pom muaj qhov tshwm sim zoo sib xws, uas yog MWCNTs thiab GONRs txo qis txoj hauv kev MITF-txog melanogenesis thiab thaum kawg txo cov ntsiab lus melanin (Daim duab 5b).

3. Kev sim cov khoom siv thiab cov txheej txheem

3.1. Kev npaj ntawm MWCNTs thiab GONRs.

Cov txheej txheem cuam tshuam rau kev ua GONRs tau tshaj tawm hauv daim ntawv dhau los.12 MWCNT (0.05 g) raug tshem tawm hauv 9:1 H2SO4/H3PO4 thiab kho nrog lub microwave reactor (CEM-Discover) nrog lub hwj chim teeb ntawm 250 W rau 2 min. Tom qab ntxiv KMnO4 (0.25 g) rau cov kev daws teeb meem, cov kev daws teeb meem tau kho nrog tib lub zog microwave ntawm 65 degree rau 4 min12 Peb mam li hloov cov txheej txheem no siv lub sij hawm luv luv thib ob ntawm microwave ntawm 8 min los ntawm kev siv lub tshuab cua compressor. Ntawm no, lub tshuab cua compressor yog siv los tswj qhov kub ntawm lub microwave reactor thaum lub sijhawm ua haujlwm. Lub zog microwave tau teeb tsa ntawm 250 W hauv kev sim ua ntej.

3.2. Kev npaj ntawm luv MWCNTs thiab luv GONRs.

Cov txheej txheem cuam tshuam rau kev ua GONRs luv luv tau tshaj tawm hauv peb daim ntawv dhau los.34 Lub sijhawm kho acidic raug xaiv li 8 teev. Lub hwj chim microwave tau teem rau ntawm 250 W, uas yog tib yam li qhov tau txais GONRs.

3.3. DPPH Radical Scavenging Activity.

DPPH tau nquag siv los txiav txim siab qhov peev txheej ntawm cov qauv thiab cov khoom siv tshuaj tiv thaiv oxidation.50 DPPH yog cov xim liab reagent uas hloov cov xim ntawm ntshav mus rau daj yog tias cov dawb radical pauv mus rau qhov ntsuas. Cov qauv tshuaj tiv thaiv oxidative zoo nrog qhov tsim nyog tau ntxiv rau cov tshuaj, thiab cov qauv raug tshuaj xyuas ntawm 517 nm rau 30 min. Peb siv cov feem pua ​​​​ntawm cov DPPH seem ntxiv nrog rau cov qauv ntsuas los ntsuas cov tshuaj tiv thaiv antioxidant uas yuav tsum tau txo qis DPPH radicals yav dhau los. Vitamin C ntawm 100 μM tau siv los ua kev tswj xyuas zoo. Qhov kev ua kom khawb (feem pua) tau ntsuas raws li

Scavenging peev (%)=(Asample − Ablank) / (Acontrol − Ablank) × 100 feem pua ​​(1)

3.4. Hlau-Chelating Activity.

Hlau ion yog qhov tseem ceeb uas ua rau lipid ntau dhau oxidation, thiab Fe2 ntxiv yog ib qho ntawm feem cuam tshuam ions.50 Qhov sib txawv ntawm nanobiomaterial (1 μL) tau ntim rau hauv 96- zoo phaj, uas muaj 2 mM FeCl2·4H2O. (10 μL), thiab tom qab ntawd muab tso rau hauv ferrozine (5 mM, 20 μL). Cov tshuaj tov tau muab sib xyaw nrog 69 μL menthol thiab khaws cia hauv chav sov li 10 min. Tom qab ntawd, cov qauv tshuaj tiv thaiv tau pom ntawm 562 nm. EDTA tau siv los ua kev tswj xyuas qhov zoo ntawm 100 μM, thiab cov qauv ntawm cov hlau-chelating kev ua ub no yog raws li eq 1.

3.5. Txo hwj chim.

Kev suav ntawm kev txo hwj chim yog raws li kev tshawb fawb yav dhau los.50 Ua ntej, 2.5 μL graphene cov ntaub ntawv yog tov nrog PBS tsis (67 mM, pH 6.8) thiab K3Fe (CN) 6 (2.5 μL, 20 feem pua) thiab tom qab ntawd incubated ntawm 50 degree rau 20 min. Tom qab ntawd, 10 feem pua ​​​​ntawm trichloro acetic acid (160 μL) tau muab sib xyaw nrog cov reagents ntawm 300g centrifuged rau 20 min. Qhov nqus tau ntev tau txiav txim siab ntawm 700 nm tom qab sib xyaw nrog 25 μL FeCl3 (2 feem pua). Butylated hydroxyanisole (BHA) tau siv ntawm 100 μM.

3.6. Kev kuaj mob qog noj ntshav.

Tib neeg dermal fibroblasts cell kab HS68 tau ua haujlwm los soj ntsuam qhov piv ntawm cell proliferation. HS68 tau tsim nyob rau hauv Dulbecco's modified Eagle medium (DMEM) uas muaj 10 feem pua ​​​​fetal bovine serum thiab 1 feem pua ​​penicillin thiab streptomycin sib xyaw.50,51 Tom qab tau kho nrog ntau qhov sib txawv ntawm cov qauv, peb tau thov MTT txhawm rau txheeb xyuas cov cell proliferation ratio. 8000 lub hlwb tau muab noob rau hauv 96- cov phiaj zoo thiab kho nrog cov qauv rau 24 teev. Cov tshuaj supernatant raug tshem tawm, thiab peb siv cov tshuaj MTT rau kab lis kev cai rau 2 teev ntawm 37 degree. Tom qab incubation, peb tshem tawm MTT-muaj xov xwm thiab yaj nws nrog dimethyl sulfoxide (DMSO). Cov tshuaj tau nyeem ntawm OD 590 nm thiab tus nqi xam los ntawm eq 1.

3.7. Kev soj ntsuam ntawm Cellular Melanin Cov ntsiab lus.

Peb siv ib txoj kev nrog kev hloov kho me me raws li qhov kev ntsuam xyuas yav dhau los.52,53 Cell pellets ntawm B16-F10 los ntawm Bioresource Collection thiab Research Center (BCRC, CRL 6323, Hsinchu, Taiwan) tau yaj hauv qhov sib xyaw ntawm 2.0. N NaOH thiab 10% DMSO. Cov qauv tom qab tau rhaub rau 1 h ntawm 90 degree thiab centrifuged ntawm 10, 000g rau lwm 10 feeb kom tau txais cov lus qhia meej meej. Kev suav melanin tau txiav txim los ntawm kev saib xyuas OD ntawm supernatant ntawm 475 nm.

3.8. B16-F10 Cellular Tyrosinase Kev Ua Haujlwm.

Rau B16-F10 cellular tyrosinase kev ua haujlwm, peb tau xa mus rau yav dhau los ua haujlwm nrog qee qhov kev hloov kho.50 Cells tau coj mus rau hauv 12-cov phiaj zoo ntawm 105 hlwb txhua qhov dej. Tom qab kev kho mob nrog cov qauv, cov hlwb tau lysed hauv 1 feem pua ​​​​Triton X-100/PBS thiab 2 mM L-tyrosine (50 μL) rau 3 teev. Tom qab incubation, peb tshem tawm cov xov xwm thiab nyeem cov absorbance ntawm OD 590 nm. Cov mis tyrosinase kev ua ub no tau suav los ntawm eq 1.

Cistanche yog tyrosinase inhibitor.

3.9. Kev kuaj pom ntawm ROS los ntawm DCFDA Staining.

Hais txog qhov kev tshawb fawb yav dhau los,54 1.2 1.105 B16-F10 hlwb tau muab noob rau hauv 6- cov phaj zoo thiab kho nrog ntau qhov ntau ntawm cov qauv. Cells raug tshem tawm hauv PBS thiab tom qab ntawd thauj khoom nrog DCFDA (5 μM) hauv cov tsis-phenol liab DMEM rau 30 min ntawm 37 degree. Lub flow cytometer (Guava, Merck, Lub teb chaws Yelemees) tau siv los txheeb xyuas cov teeb liab fluorescent ntawm DCFDA. Excitation thiab emission wavelengths ntawm DCFDA yog 488 thiab 535 nm, feem.

3.10. Quantitative Real-Time Polymerase Chain Reaction.

Peb ua raws li cov txheej txheem ntawm Lin et al. (2018).1 Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) muaj ib qho tshwj xeeb primer sojntsuam los tsim fluorescence. Nws siv cov txheej txheem fluorescence nrhiav kom pom txhua lub voj voog siv 7500 qRT-PCR System (Applied Biosystems, USA). Nws tau kuaj pom lub voj voog raws li tus nqi ntawm cov fluorescence tso tawm, thiab tom qab ntawd cov khoom ntawm txhua lub voj voog raug xam rau cov ntsiab lus tsim, uas ua rau ua tiav cov hom phiaj ntawm lub sijhawm. Trizol (Invitrogen, USA) tau siv los rho tawm RNA ua tiav ntawm lub ntsws cov ntaub so ntswg, raws li kev taw qhia los ntawm cov chaw tsim khoom. Tom qab ntawd, cov khoom siv thim rov qab (Takara, Nyiv) tau siv los tsim DNA. Nyob rau hauv qRT-PCR siv primers, teev nyob rau hauv Table 1, ua ntej, cov qauv raug rhuab los ua ib tug tib strand ntawm DNA; Tom qab ntawd ib qho kev sib khi primer coj qhov chaw los tsim ib ob-stranded DNA (dsDNA), tom qab ntawd SYBR Green dsDNA tau ua ke, uas SYBR ntsuab ntxiv cov khoom siv reagent (Roche, Basel, Swiss) tau siv, uas ua rau tso tawm fluorescence. Cov txiaj ntsig tau dhau los ntawm kev kuaj pom fluorescence. Kev tshawb pom ntawm cov teeb liab fluorescent tau tshwm sim thaum lub sijhawm elongation lossis annealing theem ntawm txhua lub voj voog; Tom qab kuaj pom, cov qauv ntsiab lus tau rov qab thawb los ntawm kev kuaj pom fluorescence intensities.55 Cov theem qhia ntawm lub hom phiaj cov noob tau normalized rau -tubulin qib siv txoj kev 2−ΔΔCt.

myricetin

3.11. Western Blot Test.

B16-F10 hlwb raug lysed ntawm 4 degree ib hmos nrog ib tug radioimmunoprecipitation assay buffer (Thermo Scientific Co., USA), uas muaj protease inhibitors. Cov khoom kuaj bicinchoninic acid protein (BCA, Sigma-Aldrich Corp., USA) tau siv los ntsuas cov protein ntau. Cov qauv protein tau muab cais tawm ntawm 10 feem pua ​​​​sodium dodecyl sulfate-polyacrylamide gel thiab xa mus rau polyvinylidene difluoride (PVDF) membrane (PALL Life Science, Ann Arbor, MI, USA). PVDF daim nyias nyias tau thaiv nrog kev thaiv tsis pub (Thermo Scientific) rau 1 h thiab incubated nrog cov thawj antibody thaum hmo ntuj ntawm 4 degree. Tom ntej no, cov membrane raug ntxuav nrog Tris buffered saline- Tween 20 tsis muaj ob zaug thiab incubated nrog lwm cov tshuaj tiv thaiv rau 1.5 teev. Tom qab ntawd, daim nyias nyias tau dipped rau hauv chemiluminescence detection reagents (Thermo Scientific) thiab soj ntsuam los ntawm MiniChemi Chemiluminescence imager (Beijing Sage Creation Science, Tuam Tshoj). Qhov chaw ntawm cov tshuaj tiv thaiv muaj xws li luav anti-MITF, luav anti-TRP-1, luav anti-TRP-2, thiab -actin (Thermo Scientific).

3.12. Kev txheeb xyuas cov khoom.

TEM (JEOL JEM-1230, 100 kV) tau siv los saib xyuas cov morphology ntawm cov nanocarbons. Lub micro Raman spectrometer (PTT, RAMaker) tau siv los xyuas cov qauv resonance ntawm nanocarbons. X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra DLD) kev ntsuas kuj tau ua tiav los txiav txim siab qhov kev tshuaj ntsuam xyuas.12,34

3.13. Kev txheeb cais.

Tag nrho cov qauv thiab cov qauv kev sim tau rov ua dua tsawg kawg peb zaug. Peb siv tus tub ntxhais kawm qhov kev xeem t-test los sib piv thiab qhia qhov nruab nrab ntawm qhov nruab nrab qhov tseem ceeb ± tus qauv sib txawv raws li kev txheeb cais.

4. Cov lus xaus

Txhawm rau ua kom tiav, peb pom tias luv GONR yog cov khoom siv rau cov khoom siv tawv nqaij vim nws ntau yam khoom siv biofunctional (Daim duab 6). Cov txiaj ntsig tau pom tias cov nanocarbons tau ua lub luag haujlwm ua cov tshuaj tiv thaiv kab mob ntxiv thiab intracellular. Lub caij no, cov nanocarbon inhibited tyrosinase kev ua thiab cov ntsiab lus melanin thiab tsis ua rau muaj kev raug mob loj rau cov xim xim. Qhov kev ua haujlwm no tau tsim los tiv thaiv melanogenesis ua haujlwm ntawm plaub hom nanocarbons; Cov kev tshawb fawb yav tom ntej yuav raug tshuaj xyuas cov txheej txheem ntawm cov tebchaw ntawm cov noob caj noob ces thiab cov protein uas muaj feem xyuam rau melanin maturation, tsheb thauj mus los, thiab tsub zuj zuj.

Cistanche txhim kho cov tawv nqaij.

Thov nias daim duab thiab tau txais cov ntsiab lus ntxiv.