Abstract:Txoj kev tshawb no tau ua los qhia meej txog qhov inhibitory cuam tshuam ntawm cycloheterophyllin ntawm melanin synthesis. Txhawm rau elucidate cov teebmeem inhibitory ntawm cycloheterophyllin ntawm B16F10 cell kab, cell viability, messenger ribonucleic acid (mRNA) qhia, tyrosinase kev ua ub no, thiab tshuaj ntsuam xyuas melanin ntau lawm. Cov teebmeem ntawm cycloheterophyllin ntawm tyrosinase-related protein 1 (TYRP1) / TYRP2 / tyrosinase (TYR) / microphthalmia-associated transcription factor (MITF) mRNA kev nthuav qhia thiab cov ntsiab lus melanin tau txiav txim siab. Quantitative real-time RT-PCR qhia tias cycloheterophyllin txo qis mRNA qhia theem ntawm TYRP1/TYRP2/TYR/MITF cov noob thiab cov ntsiab lus melanin ntau dua -MSH-kho B16F10 hlwb. Kev ntsuam xyuas kev ua tyrosinase tau qhia tias cycloheterophyllin txo qis melanin hauv B16F10 hlwb. Cov ntaub ntawv no qhia tau hais tias cycloheterophyllin nce cov nyhuv whitening hauv B16F10 hlwb; Yog li, cycloheterophyllin yog ib qho khoom muaj zog rau cov tawv nqaij dawb. Yog li, kev tshawb fawb ntxiv ntawm cov txheej txheem ntawm kev ua ntawm cycloheterophyllin rau kev txhim kho cov khoom siv ua haujlwm yuav tsum tau tshawb xyuas.

Raws li cov kev tshawb fawb cuam tshuam, cistanche yog ib hom tshuaj ntsuab uas hu ua "cov tshuaj ntsuab muaj txuj ci uas ua rau lub neej ntev". Nws lub ntsiab tivthaiv yog cistanoside, uas muaj ntau yam teebmeem xws li antioxidant, tiv thaiv kev mob, thiab kev tiv thaiv kev ua haujlwm. Cov txheej txheem ntawm cistanche thiab tawv nqaij dawb yog nyob rau hauv cov nyhuv antioxidant ntawm cistanche glycosides. Melanin nyob rau hauv tib neeg daim tawv nqaij yog tsim los ntawm oxidation ntawm tyrosine catalyzed los ntawm tyrosinase, thiab cov tshuaj tiv thaiv oxidation yuav tsum muaj kev koom tes ntawm oxygen, yog li cov pa-dawb radicals nyob rau hauv lub cev ua ib qho tseem ceeb cuam tshuam rau melanin ntau lawm. Cistanche muaj cistanoside, uas yog ib qho antioxidant thiab tuaj yeem txo qhov tsim ntawm cov dawb radicals hauv lub cev, yog li inhibiting melanin ntau lawm.

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1. Taw qhia

Cov xim ntawm daim tawv nqaij yog txiav txim los ntawm hemoglobin nyob rau hauv cov ntshav, melanin pigment nyob rau hauv lub epidermal txheej, thiab -carotene nyob rau hauv cov ntaub so ntswg subcutaneous. Ntawm lawv, cov xim ntawm daim tawv nqaij yog txiav txim siab los ntawm tus nqi thiab kev faib tawm ntawm cov xim melanin [1]. Melanin yog cov xim uas tsim los ntawm melanosome nyob rau hauv melanocyte uas muaj nyob rau hauv basal txheej. Nws absorbs los yog scatters ultraviolet (UV) rays thiab muaj cov txiaj ntsig zoo los tiv thaiv kev puas tsuaj rau daim tawv nqaij lossis cov ntaub so ntswg ntawm daim tawv nqaij. Txawm li cas los xij, ntau dhau ntawm melanin tuaj yeem ua rau melanin, freckles, thiab pigmentation. Nws paub tias ua rau hyperpigmentation [2,3].

Melanin pigment yog ib tug sawv cev irritant uas ua rau exogenous laus. Nws yog tsim los ntawm melanogenesis ntawm melanocytes uas tau raug UV rays thiab muaj txiaj ntsig zoo ntawm kev tiv thaiv daim tawv nqaij los ntawm UV rays los ntawm kev ua kom tawv nqaij xim. Cov xim melanin muaj eumelanin (dub) thiab pheomelanin (liab-xim av) [4,5]. Melanin yog tsim los ntawm kev siv L-tyrosine, ib hom amino acid, ua substrate, thiab los ntawm L-DOPA (3, 4-dihydroxyphenylalanine) thiab ntxiv oxidized rau DOPA-quinone los ntawm lub ntsiab enzymes tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), thiab TYRP2. DOPA-quinone yog ntxiv oxidized rau liab-daj pheomelanin nyob rau hauv lub xub ntiag ntawm sulfhydryl pawg. DOPA-quinone kuj yog decarboxylated, oxidized, thiab catalyzed rau hauv eumelanin thaum sulfhydryl pawg depleted [4,5]. Tyrosinase enzyme yog lub luag haujlwm tseem ceeb rau kev tsim cov melanin, thiab nws paub tias licorice extract, Japanese oak extract, arbutin, thiab kojic acid, uas yog cov sawv cev ntawm cov ntaub ntawv dawb, inhibit qhov kev ua ntawm tyrosinase enzyme [6,7]. Tshwj xeeb, arbutin plays lub luag haujlwm hauv inhibiting cov haujlwm ntawm tyrosinase los ntawm kev ua haujlwm sib tw nrog L-tyrosine, uas yog cov khoom siv raw rau melanin, thiab kojic acid khi rau qhov chaw nquag ntawm tyrosinase los ntawm Cu2 ntxiv. Kojic acid thiab arbutin paub tias muaj zog ua kom tawv nqaij, tab sis cov kev mob tshwm sim ntawm cov teeb meem kev nyab xeeb xws li khaus tawv nqaij tau raug tshaj tawm [8,9]. Ntau qhov kev tshawb fawb tau ua los tshawb pom cov khoom siv tau zoo tshiab xws li kev tsim cov khoom siv ua haujlwm tau zoo [8,9].

Cycloheterophyllin (C30H30O7), ib hom prenylflavone, muaj ntau muaj nyob rau hauv Artocarpus heterophyllus thiab tau raug tshaj tawm tias muaj tshuaj thiab biological functions xws li tshuaj tiv thaiv kab mob, anti-platelet kev ua, thiab antioxidant kev ua tau zoo [10–13]. Tsis tas li ntawd, nws tau raug tshaj tawm tias nws muaj cov tshuaj tiv thaiv antioxidant thiab tiv thaiv kev laus ntawm tib neeg dermal fibroblasts vim UV-A [14,15].

Yog li ntawd, nws tau raug tshaj tawm tias cycloheterophyllin muaj txiaj ntsig zoo hauv ntau yam kab mob, tab sis tsis muaj kev tshawb fawb txog kev tsim cov tshuaj melanin los ntawm cycloheterophyllin. Hauv txoj kev tshawb no, kuv tau tshuaj xyuas qhov cuam tshuam ntawm cycloheterophyllin ntawm kev qhia ntawm cov cim TYRP1, TYRP2, TYR, thiab MITF hauv B16F10 hlwb, cov kab mob melanoma tau los ntawm cov nas, cuam tshuam nrog cov txheej txheem tsim melanin.

2. Cov txiaj ntsig thiab kev sib tham

CCK{0}} kev soj ntsuam tau ua los txiav txim siab cytotoxicity ntawm cycloheterophyllin hauv B16F10 cell kab. Ntau qhov ntau ntawm cycloheterophyllin tau ntxiv los ntsuas kev muaj peev xwm ntawm tes (Daim duab 1). Thaum kho nrog qhov concentration ntawm 20 µg / mL lossis ntau dua, nws tau lees paub tias qhov muaj sia nyob ntawm B16F10 hlwb poob qis dua piv rau pawg tswj hwm. Thaum cycloheterophyllin ntxiv nrog rau qhov concentration ntawm 10 µg / mL, qhov ciaj sia taus ntawm B16F10 hlwb zoo ib yam li pawg tswj hwm. Yog li cycloheterophyllin tau ntxiv ntawm qhov ntau ntawm 10, 1, thiab 0.1 µg / mL hauv kev sim ntxiv.

Tyrosinase enzyme reacts nrog L-tyrosine los tsim L-DOPA, thiab L-DOPA thaum kawg tsim cov xim melanin los ntawm DOPAquinone los ntawm tyrosinase, uas yog lub npe hu ua ib qho tseem ceeb enzyme uas tswj melanogenesis [4,5]. Hauv qhov kev sim no, kojic acid tau siv los ua kev tswj hwm zoo los ntsuas qhov qib ntawm inhibition ntawm tyrosinase enzyme kev ua haujlwm. Qib ntawm inhibition ntawm tyrosinase enzyme kev ua haujlwm tau lees paub los ntawm kev kho mob nrog cycloheterophyllin ntawm qhov siab ntawm 10, 1, thiab 0.1 µg/mL. Nws tau raug lees paub tias tyrosinase enzyme kev ua haujlwm tau inhibited nyob rau hauv ib qho concentration-dependent yam los ntawm cycloheterophyllin (Daim duab 2). Hauv pawg cycloheterophyllin-kho ntawm qhov ntau ntawm 10, 1, thiab 0.1 µg / mL, nws tau lees paub tias qhov cuam tshuam ntawm tyrosinase enzyme yog 58.0, 27.4, thiab 16.9 feem pua.

UV rays nkoos keratinocytes nyob rau hauv lub epidermis kom induce qhia ntawm - melanocyte-stimulating hormone (-MSH), ib tug hormone uas nkoos melanocytes, thiab yog secreted sab nraum lub cell, thiab secreted -MSH stimulates melanocytes. Los ntawm kev khi rau melanocortin 1 receptor (MC1R), nws ua rau muaj kev cuam tshuam rau melanin synthesis hauv cell, ua rau cov xim melanin ntau lawm [5]. Hauv qhov kev sim no, kom induce melanin ntau lawm hauv B16F10 hlwb, 200 nM -MSH tau siv los ntxias melanin ntau lawm hauv B16F10 hlwb [16]. Real-time RT-PCR ntawm kev qhia theem ntawm TYRP1, TYRP2, TYR, thiab MITF cov noob, uas yog cov cim uas tau nce los ntawm -MSH kev kho mob tau txiav txim siab, los ntawm kev kho cycloheterophyllin hauv -MSH-kho B16F10 hlwb. B16F10 hlwb tau kho nrog cycloheterophyllin thiab kev qhia ntawm tus cim tau lees paub los ntawm lub sijhawm RT-PCR. Raws li qhov tshwm sim, nws tau lees paub tias cov lus qhia ntawm TYRP1, TYRP2, TYR, thiab MITF tau txo qis hauv pawg neeg kho cycloheterophyllin piv rau pawg -MSH-kho. Tshwj xeeb, hauv pawg sim kho nrog 10 µg / mL ntawm cycloheterophyllin, kev qhia ntawm TYRP1, TYRP2, TYR, thiab MITF cov noob tau txo qis los ntawm 53.3, 26.6, 58, thiab 34 feem pua, raws li (Daim duab 3). Hauv pab pawg sim kho nrog 1 µg / mL cycloheterophyllin, nws tau lees paub tias qhov kev qhia ntawm cov cim tau txo qis, thiab kev qhia ntawm TYRP1 thiab TYRP2 noob tau zoo sib xws hauv 0.1 µg / mL cycloheterophyllin-kho pawg.

Tom qab lees paub tias kev kho mob ntawm cycloheterophyllin txo qis mRNA qhia theem ntawm tus cim ntsig txog melanogenesis, tus nqi ntawm melanin pigment ntau lawm tau lees paub los txiav txim seb puas tsim cov xim melanin hauv B16F10 hlwb tau txo qis (Daim duab 4) . B16F10 cell kab kho nrog cycloheterophyllin ntawm qhov ntau ntawm 10, 1, thiab 0.1 µg / mL, raws li tau sau tseg, thiab qib ntawm melanin ntau lawm tau lees paub. Raws li qhov tshwm sim, nws tau lees paub tias kev tsim cov xim melanin tau txo qis los ntawm cycloheterophyllin ntawm qhov ntau ntawm 10 thiab 1 μg / mL (Daim duab 4A). Tsis tas li ntawd, nws tau lees paub tias kev tsim cov xim melanin hauv cov mob cycloheterophyllin-kho tau txo qis piv rau pawg tswj hwm (Daim duab 4B). Thaum 10 thiab 1 µg / mL cycloheterophyllin tau ntxiv, qhov tsim tawm ntawm cov xim melanin poob los ntawm 44.6 thiab 37.8 feem pua, raws li, uas tau txo qis dua piv rau pawg tswj hwm (Daim duab 4B). Cov txiaj ntsig no zoo ib yam nrog cov txiaj ntsig kev sim ntawm lub sijhawm tiag tiag RT-PCR ntawm mRNA qhia qib ntawm cov cim qhia txog melanogenesis.

Antioxidants xws li ascorbic acid paub tias yog ib qho tseem ceeb ntawm cov khoom siv dawb uas inhibits cov synthesis ntawm melanin pigments los ntawm kev txo DOPAquinone, ib qho khoom nruab nrab thaum lub sij hawm eumelanin ntau lawm txheej txheem, rau L-DOPA [16,17]. Cycloheterophyllin kuj tau tshaj tawm tias muaj cov teebmeem antioxidant [11,12]. Nws tau pom tias cycloheterophyllin tuaj yeem ua tau zoo hauv kev ntxuav hniav dawb rau cov nyhuv antioxidant. Raws li qhov tshwm sim ntawm kev lees paub cov nyhuv antioxidant hauv vitro los ntawm qhov kev ntsuam xyuas DPPH radical scavenging, nws tau lees paub tias nws muaj cov nyhuv antioxidant piv rau ascorbic acid, kev tswj zoo nyob rau hauv siab concentration ntawm cycloheterophyllin (Daim duab 5). Nyob rau hauv lub neej yav tom ntej, kev tshawb fawb ntxiv txog cov tshuaj tiv thaiv oxidative ntawm cycloheterophyllin hauv melanocytes thiab qhia txog cov tshuaj tiv thaiv oxidative xws li catalase, SOD1, SOD2, thiab SOD3 yuav tsum tau ua.

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Kev sim cov ntaub ntawv thiab Cell Culture

B16F10 xov tooj ntawm tes tau yuav los ntawm Kauslim Cell Line Bank (Korea), thiab cov kab lis kev cai ntawm tes nruab nrab yog 10 feem pua ​​​​fetal bovine serum (FBS, SFBU-0500, Equitech-bio, Kerrville, TX, USA) hauv Dulbecco's modified Eagle's medium (DMEM , LM 001–05, Welgene, Gyeongsan-si, Kauslim), thiab 1 feem pua ​​penicillin/streptomycin (Gibco, 15140–122, Waltham, MA, USA) tau ntxiv. Cells tau coj mus rau hauv lub incubator ntawm 37 ◦C thiab 5 feem pua ​​CO2 [16]. Lub cycloheterophyllin siv nyob rau hauv qhov kev sim no tau muab rho tawm los ntawm Artocarpus heterophyllus siv DMSO thiab yuav los ntawm ChemFaces Co. (CFN97748, Wuhan, Tuam Tshoj, Purity Ntau dua lossis sib npaug li 98 feem pua).

3.2. Cell Viability Assay Cell

Kev vam meej tau raug soj ntsuam los ntawm CCK-8 daws (Cell suav cov khoom siv; EZ−3000, EZ-Cytox, Seoul, DoGen). B16F10 cov kab xov tooj ntawm tes tau cog rau ntawm kab lis kev cai nruab nrab ntawm qhov ntom ntawm 2 × 104 hlwb / 96 zoo daim hlau. Tom qab 24 teev cycloheterophyllin (CFN97748, Chemfaces) tau ntxiv. B16F10 cell kab kho nrog cycloheterophyllin yog incubated rau 24 teev thiab CCK{13}} tov tau ntxiv rau 30 min nyob rau hauv 37 ◦C incubator. Absorbance tau ntsuas ntawm 450 nm siv lub microplate nyeem ntawv (Epoch, Winooski, VT, USA, BioTek) thiab cell viability raug xam raws li absorbance ntawm pawg tswj tsis muaj hlwb [16].

3.3. DPPH Assay

3.4. Hauv Vitro Mushroom Tyrosinase Kev Ua Haujlwm

3.5. Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (Q-RT-PCR)

Tag nrho RNA raug cais tawm siv Trizol (15596018, Carlsbad, CA, USA, Thermo Fisher Scientific). Qhov concentration ntawm RNA tau txiav txim siab spectrophotometrically (BioTek). Plaub micrograms ntawm RNA tau thim rov qab rau hauv cDNA siv ReverTra Ace® rov qab cov khoom siv (Toyobo, FSQ101, Osaka, Nyiv). Qhov rov qab hloov pauv tau raug tso tseg los ntawm kev ntxiv Tris-EDTA tsis kam (pH 8.0) rau tag nrho 200 µL ntawm cDNA tov. Cov TaqMan® Gene Expression Assay teeb tau yuav los ntawm Applied Biosystems. Q-RT-PCRs tau ua tiav raws li cov chaw tsim khoom cov lus qhia. Luv luv, 20 µL ntawm Q-PCR sib tov muaj 10 µL 2 × TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), 1 µL 20 × Taqman expression assay (Applied Biosystems), thiab 50 ng cDNA. Q-PCR sib tov tau tshwm sim ntawm 95 ◦C rau 20 s thiab 40 zaug cycling cov tshuaj tiv thaiv (95 ◦C rau 1 s thiab 60 ◦C rau 20 s) nrog rau StepOnePlus System (Applied Biosystems). Cov lej txheeb xyuas cov noob rau TaqMan qhia kev ntsuas siv hauv Q-RT-PCR kev tshuaj ntsuam tau nthuav tawm hauv Table 1. Tib neeg GAPDH (Applied Biosystems) tau siv rau normalizing variation hauv cDNA ntau los ntawm cov qauv sib txawv.

3.6. Melanin Contest Assay

Txhawm rau ntsuas tus nqi melanin pigment, Hosoi txoj kev yog ib feem ntawm kev hloov pauv [17]. Tag nrho ntawm 1 × 105 B16F10 hlwb raug inoculated rau hauv 60 mm kab lis kev cai tais. Tom qab culturing lub hlwb rau 24 h, 200 nM ntawm -melanocyte stimulating hormone (-MSH; M4135, Sigma-Aldrich) thiab cycloheterophyllin (10, 1, 0.1 µg / mL) tau ntxiv rau 72 h ntawm txhua qhov concentration. Tom qab tshem tawm cov kab lis kev cai nruab nrab, 200 µL ntawm NaOH (1N; S8045, Sigma-Aldrich) tov tau ntxiv thiab incubated ntawm 60 ◦C rau 2 h kom yaj melanin. Lub absorbance yog ntsuas nrog ib tug spectrophotometer (405 nm, Epoch). Raws li kev tswj hwm zoo, 100 µg / mL arbutin (A4256, Sigma-Aldrich) tau siv. Inhibition ntawm zus tau tej cov xim melanin tau qhia raws li ib feem pua ​​ntawm cov melanin uas tsim nyob rau hauv-MSH kev kho mob tej yam kev mob. Nws tau ua los ntawm nws tus kheej ua peb qhov kev sim ywj pheej.

3.7. Kev txheeb cais

4. Cov lus xaus

UV ntsib nyob rau hauv lub neej niaj hnub muaj txiaj ntsig zoo xws li vitamin D synthesis thiab sterilization, tab sis nyob rau hauv lwm yam, nws ua rau poob ntawm daim tawv nqaij elasticity, wrinkles, pigmentation, erythema, o, thiab lwm yam, los ntawm ib tug saw cov tshuaj tiv thaiv ntawm reactive oxygen hom [18, 19] ib. Tsis tas li ntawd, exogenous aging tshwm sim los ntawm lwm yam xws li UV tiv thaiv kev ua haujlwm ntawm cov ntaub so ntswg ib txwm muaj thiab rov tsim dua tshiab thaum cov ntaub so ntswg puas vim qhov txo qis thiab tsis zoo ntawm cov khoom siv hauv lub cev [20,21].

Los ntawm txoj kev tshawb no, nws muaj peev xwm paub meej tias qhov tsim nyog concentration ntawm cycloheterophyllin los kho B16F10 hlwb. Nws tau raug lees paub tias qhov concentration ntawm 10 µg / mL tsis cuam tshuam rau kev muaj peev xwm ntawm cov hlwb, raws li qhia hauv daim duab 1. Los ntawm kev ntsuas cov txiaj ntsig ntawm kev ua haujlwm inhibitory nyhuv ntawm tyrosinase enzyme, nws tau lees paub tias cycloheterophyllin concentrations ntawm 10, 1, thiab 0.1 µg / mL txo cov haujlwm ntawm tyrosinase enzyme (Daim duab 2). Tsis tas li ntawd, nws tau lees paub tias cycloheterophyllin txo qis kev qhia ntawm cov cim (TYRP1, TYRP2, TYR, thiab MITF gene) piv rau -MSH kho mob (Daim duab 3). Tsis tas li ntawd, nws tau lees paub tias cycloheterophyllin txo qis melanin synthesis hauv B16F10 hlwb (Daim duab 4). Raws li cov txiaj ntsig no, cycloheterophyllin qhia tau tias muaj peev xwm ua tus neeg sib tw ua kom tawv nqaij tshiab.

Txoj kev tshawb no yog thawj zaug kom paub meej tias cov nyhuv inhibitory ntawm cycloheterophyllin ntawm melanin synthesis thiab qhia txog qhov muaj peev xwm ntawm inhibiting tawv nqaij laus los ntawm UV rays. Tsis tas li ntawd, ntxiv rau qhov tob ntawm nws cov teebmeem ntawm cov teeb liab mechanisms ntawm B16F10 hlwb, nyob rau hauv lub signaling systems koom nyob rau hauv whitening, thiab nyob rau hauv ntau yam antioxidant enzymes nyob rau hauv melanocytes zoo li yuav tsum tau.

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