Cyclopentanone Exerts Anti-Melanogenesis Thiab Anti-Wrinkle Activities hauv B16F10 Melanoma

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

Hee Jin Jung 1, A Kyoung Lee 1,2, Yeo Jin Park 1,2, Sanggwon Lee 1,2, Dongwan Kang 1,2, Young Suk Jung 1,2, Hae Young Chung 1,2 thiab Hyung Ryong Moon 1, 2, *

Abstract:Ultraviolet (UV) hluav taws xob raug yog thawj qhov ua rau extrinsic tawv nqaij laus, uas ua rau cov tawv nqaij hyperpigmentation thiab wrinkling. Hauv txoj kev tshawb no, peb tau tshawb xyuas cov nyhuv whitening ntawm (2E,5E)-2,5-bis(3-hydroxy-4-}methoxybenzylidene)cyclopentanone (BHCP) onB16F10 melanoma thiab nws cov tshuaj tiv thaiv. wrinkle kev ua haujlwm ntawm Hs27 fibroblasts hlwb. BHCP tau pom tias muaj zog tiv thaiv tyrosinase, nrog 50 feem pua ​​​​ inhibition concentration (IC50) qhov tseem ceeb ntawm 1.10 μM thiab 8.18 μM formycophenolate (L-tyrosine) thiab diphenolase (L-DOPA), thiab kev tshawb fawb enzyme kinetics qhia tias BHCP yog ib qho kev sib tw-hom inhibitor tyrosina. . Tsis tas li ntawd, BHCP cuam tshuam cov ntsiab lus melanin thiab cellular tyrosinase kev ua haujlwm thiab txo qis cov qib ntawm microphthalmia-associatedtranscription factor (MITF), phosphorylated qib ntawm cAMP teb cov ntsiab lus-binding (CREB) protein, thiab tyrosinase hauv -melanocyte-stimulating hormone (-MSH)-induced. B16F10 melanoma hlwb. Ntxiv mus, BHCP inhibited phosphorylation ntawm p65 thiab kev qhia ntawm matrix metalloproteinases (MMP-1, MMP-9, MMP-12, thiab MMP-13) hauv Hs27 fibroblasts stimulated nrog UV hluav taws xob.Yog li ntawd, peb cov txiaj ntsig tau pom tias BHCP tuaj yeem yog tus neeg sib tw zoo rau kev txhim kho cov tshuaj kho mob rau cov kab mob cuam tshuam nrog hyperpigmentation thiab wrinkling.

Cov ntsiab lus: (2E,5E)-2,5-Bis(3-hydroxy-4-methoxybenzylidene)cyclopentanone (BHCP); tyrosinase inhibitor; anti-melanogenesis; anti-wrinkle

Cistanche yoganti-wrinkling.

1. Taw qhia

Kev laus ntawm daim tawv nqaij yog cov txheej txheem nyuaj thiab txhim kho uas ua rau kev ua haujlwm zoo thiab zoo nkauj hloov hauv daim tawv nqaij, nrog rau ob qho tib si sab hauv thiab sab nrauv yog lub luag haujlwm [1]. Extrinsic tawv nqaij laus yog tshwm sim los ntawm ib puag ncig aggressors, xws li ultraviolet (UV) hluav taws xob, kev nyuaj siab, los yog haus luam yeeb. Txawm li cas los xij, nws feem ntau tshwm sim los ntawm rov raug UV los ntawm lub hnub, uas yog hu ua photoaging. Daim tawv nqaij photoaging yog yam ntxwv los ntawm ntxhib thiab sib sib zog nqus wrinkles, thickness, roughness, dyspigmentation, thiab histological hloov [2-4].

Tyrosinase (EC 1.14.18.1), uas tseem hu ua polyphenol oxidase, yog ib qho ntawm cov enzymes multifunctionalcopper-muaj cov enzymes koom nrog hauv melanin synthesis thiab pom dav dav hauv xwm [5].Tyrosinase feem ntau muaj nyob rau hauv feem ntau ntawm cov kab mob, cov nroj tsuag, thiab tsiaj. Hauv cov nroj tsuag, tyrosinase ua los ntawm oxidizing monophenols rau hauv diphenols (mycophenolate kev ua si) thiab koom nrog oxidation ntawm o-diphenols rau hauv o-quinones (diphenolase kev ua), tom qab ntawd los ntawm oxidation ofquinones rau hauv cov xim av tsaus nti [6].

Melanogenesis yog kev hloov pauv ntawm L-tyrosine rau hauv 3, 4-dihydroxyphenylalanine (L-DOPA), uas L-DOPA hloov mus rau DOPA quinine [7]. Yog li, tyrosinase plays lub luag haujlwm tseem ceeb hauv kev tsim melanin hauv melanocytes, thiab inhibition ntawm tyrosinase yog lub hom phiaj zoo nkauj rau kev txhim kho cov pigmentation-txog kev mob thiab rau kev loj hlob ntawm whiteningagents [8,9]. Melanin synthesis yog induced los ntawm ntau yam stimuli, xws li UV thiab tshuaj xws liisobutylmethylxanthine (IBMX) thiab alpha-melanocyte-stimulating hormone (-MSH). -MSH khi rau nws cov receptor melanocortin 1 receptor (MC1R), tom qab nce qib cytoplasmic cyclic AMP (cAMP). Kev nce qib cAMP ua rau muaj protein ntau kinase A (PKA), uas ua rau kev nthuav tawm ntawm microphthalmia-txuas nrog kev hloov pauv hloov pauv (MITF) los ntawm phosphorylation ntawm cAMP cov lus teb cov ntsiab lus-binding protein (CREB). MITF induces kev qhia ntawm tyrosinase, tyrosinase-related protein (TRP)-1, thiab TRP{19}}, uas thaum kawg ua rau muaj zog melanin synthesis [10]. MITF suav hais tias yog ib qho tseem ceeb ntawm kev hloov pauv ntawm melanogenesis; Yog li ntawd, ntau cov kev tshawb fawb tau ua los tswj qhov kev nthuav qhia ntawm MITF los inhibit melanogenesis [11].

Wrinkle tsim yog paub tias muaj kev sib raug zoo nrog kev degradation ntawm extracellularmatrix ntawm daim tawv nqaij, thiab UV hluav taws xob activates nuclear factor-κB (NF-κB), yog li ua rau zus tau tej cov collagen fragmentation thiab matrix metalloproteinases (MMPs) [2]. MMPs yog zinc-dependent endopeptidases uas yog ib qho tseem ceeb nyob rau hauv kev kho ntawm extracellular matrixstructure nyob rau hauv daim tawv nqaij. Yog li ntawd, ntau dhau degradation ntawm collagen thiab matrix los ntawm UV-inducedMMPs yog ib tug yam ntxwv feature ntawm photodamaged daim tawv nqaij, thiab MMPs yog siv raws li ib tug tseem ceeb cim ntawm UV-induced photoaging raws li zoo raws li daim tawv nqaij mob [12].

Cov curcumin zoo li diarylheptanoid skeleton derivatives muaj xws li antioxidants, reportanticancer, activities.scaffolds, nrog rau (2E,5E)-2,5-bis(3-hydroxy-4-methoxybenzylidene)cyclopentanone (BHCP) (Daim duab 1),kom nthuav tawm ntau yam kev tiv thaiv kab mob, tiv thaiv melanogenesis, tshwj xeeb tshaj yog, Leow li al. [19] qhia nyob rau hauv bioactivities, thiab anti-tyrosinase [13–18] dibenzylidene-cyclopentanone 2014 uas yuav ua rau muaj kev tiv thaiv los ntawm tib neeg osteosarcoma los ntawm kev tswj ntawm Wnt / -cateninsignaling txoj kev. Txawm li cas los xij, cov tshuaj tiv thaiv melanogenesis thiab kev tiv thaiv wrinkle ntawm BHCP tseem tab tom nrhiav pom, thiab cov txheej txheem molecular hauv qab nws cov haujlwm tseem tsis tau tsim kom meej meej.

Daim duab 1. Cov qauv ntawm (2E,5E)-bis(3-hydroxy-4-methoxybenzylidene)cyclopentanone (BHCP).

Hauv txoj kev tshawb fawb tam sim no, peb tau tshawb xyuas cov tshuaj tiv thaiv melanogenesis thiab kev tiv thaiv kev puas tsuaj ntawm BHCP, thiab nrhiav kev txheeb xyuas cov txheej txheem cuam tshuam, tshwj xeeb tshaj yog hais txog cov ntsiab lus melanin thiab cellular tyrosinase kev ua, uas tau tshawb fawb siv tyrosinase inhibition assay thiab tsom xam ntawm enzyme kinetics. Ntxiv mus, peb tau pom tias BHCP ua rau muaj kev cuam tshuam rau melanogenesis thiab wrinkles uas cuam tshuam nrog kev txo qis ntawm CREB / MITF / tyrosinase hauv -MSH-inducedB16F10 nas melanoma hlwb thiab inhibition ntawm phosphorylation ntawm p65 thiab MMP qhia hauv UV-induced Hs27 human fibroblast.

zaj tshuaj ntsuab cistanche

2. Cov txiaj ntsig thiab kev sib tham

2.1. Synthesis ntawm (2E,5E)-2,5-Bis(3-hydroxy-4-}methoxybenzylidene)cyclopentanone (BHCP)

Kev daws ntawm {{0}}hydroxy-4-methoxybenzaldehyde (100 mg, 0.66 mmol, isovanillin) andcyclopentanone (0.03 mL, 0.33) mmol) hauv 1 N HCl-acetic acid tov (0.02 mL) tau nplawm ntawm 25 ◦C rau 2 h. Tom qab sawv ntsug rau 1 hnub, cov tshuaj tiv thaiv sib tov tau kho nrog dej txias hauv qhov muaj methanol methanol, lim, thiab ntxuav nrog dej txias kom tsim tau BHCP (65.9 mg) hauv 56.9 feem pua ​​​​yield.BHCP tau txheeb xyuas los ntawm spectroscopic txoj kev, suav nrog 1H thiab 13C-NMR, nrog rau kev sib piv nrog cov ntaub ntawv luam tawm spectral thiab Thin Layer Chromatography (TLC) tsom xam [19]. Cov qauv yog qhia hauv daim duab 1.

BHCP: daj amorphous hmoov (CHCl3); 1H-NMR (500 MHz, DMSO-d6) δ: 9.25 (s, 2H, 2 × OH), 7.28 (s, 2H, 2 × vinylicH), 7.14 (d, 2H , J=2 . H), 7.01 (d, 2H, J=8.5 Hz, 2 × 5-H), 3.81 (s, 6H, 2 × OMe), 3.01 (s, 4H, 2 × CH2) ; 13C-NMR (100 MHz, DMSO-d6) δ: 195.5, 149.9, 147.2, 136.0, 133.1, 129.1, 124.3, 117.5, 112.8, 56.3, 26.6; ESI-MS: m/z 351 (M − H)-.

2.2. Inhibitory nyhuv ntawm BHCP ntawm Mushroom Tyrosinase Activity

Raws li pom nyob rau hauv Table 1, BHCP inhibited tyrosinase nrog IC50 qhov tseem ceeb ntawm 1.10 ± 0.12 μMand 8.18 ± 0.44 μM, whereas kojic acid (zoo tswj) muaj IC50 qhov tseem ceeb ntawm 18.68 μ 1.8 ± 1.40 Mand μM rau monophenolase thiab diphenolase, feem. Hauv peb txoj kev tshawb fawb yav dhau los ntawm cov tshuaj tua hluav taws xob muaj peev xwm ntawm tyrosinase, peb pom cov txheej txheem los ntawm cov qauv molecular kev tshawb fawb uas cov 3-hydroxy thiab 4-methoxy pawg ntawm benzylidene muaj kev sib khi zoo heev rau ntawm tyrosinase active site [20]. Los ntawm qhov kev tshawb fawb no, nws tau pom tias nws cov pab pawg ua haujlwm (3-hydroxy thiab 4- pawg methoxy) tau cuam tshuam nrog kev nce ntxiv hauv tyrosinaseinhibitory kev ua.

Table 1. Tyrosinase inhibitory kev ua si thiab enzyme kinetic tsom xam ntawm BHCP.

Tsis tas li ntawd, lub luag haujlwm ntawm tyrosinase inhibition los ntawm BHCP tau tshawb xyuas los ntawm enzyme kinetic tsom xam hauv txoj kev tshawb no (Table 1 thiab daim duab 2). Lineweaver-Burk cov phiaj xwm tau rub tawm cov ntaub ntawv tau los ntawm kev tshawb fawb kinetic, thiab qhov inhibition tas li (Ki) tau txais los ntawm Dixon cov phiaj xwm. Cov Lineweaver-Burk ob chav sib txuas lus qhia tau hais tias muaj kev sib tw-hom inhibition.Raws li pom hauv daim duab 2a-c, BHCP ua raws li kev sib tw inhibitor ntawm L-tyrosine thiab L-DOPA. Tsis tas li ntawd, cuam tshuam ntawm x-axis ntawm Dixon cov phiaj xwm feem ntau. siv los txiav txim seb hom enzyme inhibition tas li (Ki) rau ib qho enzyme-inhibitor complex [21,22], qhov twg tus nqi ntawm lub x-axis qhia tus nqi ntawm −Ki. Raws li tau piav qhia hauv daim duab 2b-d, Ki qhov tseem ceeb ntawm BHCP yog 1.7 μMand 10.5 μM raws li cov substrates rau L-tyrosine thiab L-DOPA, feem. Raws li tus nqi Ki sawv cev rau qhov kev xav tau los tsim ib qho enzyme-inhibitor complex, tus nqi Ki qis dua qhia tias muaj txiaj ntsig zoo dua tiv thaiv tyrosinase.

Daim duab 2. Lineweaver–Burk (a,c) thiab Dixon (b,d) plots rau tyrosinase enzyme inhibition los ntawm BHCP.

2.3. Qhov cuam tshuam ntawm BHCP ntawm Cell Viability ntawm B16F10 Melanoma thiab Hs27 Fibroblast Cells

Ua ntej txiav txim siab seb BHCP tau siv cov tshuaj tiv thaiv melanogenesis thiab tiv thaiv kev ua ub ua no, peb tau tshuaj xyuas cytotoxicity ntawm BHCP rau B16F10 hlwb thiab Hs27 hlwb los ntawm kev kho nrog sib txawv ntawm BHCP rau lub sijhawm sib txawv, thiab kev ua haujlwm ntawm tes tau ntsuas nrog EZ-Cytox assay. Raws li pom nyob rau hauv daim duab 3a, b, mus txog 10 μM ntawm BHCP rau 48 h tsis txo cov ciaj sia ntawm B16F10 hlwb lossis Hs27 hlwb. Tom qab ntawd, kev tshawb fawb ntxiv hauv vitro ntawm kev tiv thaiv melanogenesis thiab kev ua haujlwm ntawm BHCP tau ua nrog 1, 5, thiab 10 μM.

2.4. Inhibition ntawm BHCP tawm tsam Melanin Cov ntsiab lus thiab Cellular Tyrosinase Kev Ua Haujlwm hauv B16F10 Melanoma Cells

Txhawm rau txiav txim siab seb BHCP puas muaj peev xwm inhibitory ntawm cov ntsiab lus melanin ntawm -MSH-inducedB16F10 hlwb, cov hlwb raug pretreated nrog cov qhia sib txawv (1, 5, thiab 10 μM) ntawm BHCP los yog kojic acid (5 mM) rau 24 h thiab ces stimulated. nrog -MSH rau 48 teev. Raws li pom hauv daim duab 4a, cov ntsiab lus melanin hauv cov hlwb kho nrog BHCP nyob rau hauv lub xub ntiag ntawm -MSH txo qis concentration-dependent yam, qhia 113 feem pua ​​​​ntawm 1 μM, 106 feem pua ​​​​ntawm 5 μM, thiab 102 feem pua ​​​​ntawm 10 μM, piv rau tswj pab pawg kho nrog -MSH nkaus xwb (186 feem pua). Qhov zoo siab, BHCP (1 μM) inhibited melanin cov ntsiab lus ntau dua kojic acid (5 mM). Tsis tas li ntawd, kev kuaj mob ntawm tes tyrosinase tau ua los ntsuas cov nyhuv inhibitory ntawm BHCP ntawm B16F10 hlwb. Raws li pom nyob rau hauv daim duab 4b, BHCP poob nyob rau hauv ib tug concentration-dependent yam nrog lub tyrosinase kev ua si los ntawm 120 feem pua ​​​​ntawm 1 μM, 116 feem pua ​​​​ntawm 5 μM, thiab 105 feem pua ​​​​ntawm 10 μM, piv rau pawg tswj kev kho nrog -MSH nkaus xwb (181 feem pua ​​) Cov nyhuv inhibitory ntawm BHCP muaj zog ntau dua li ntawm kojic acid; Qhov inhibition ntawm BHCP ntawm 1 μM yog qhov zoo tshaj ntawm kojic acid ntawm 5 mM (131 feem pua). Cov txiaj ntsig no qhia tias BHCP muaj cov txiaj ntsig zoo los ntawm inhibiting melanin biosynthesis thiab intracellular tyrosinase synthesis hauv B16F10 melanocytes.

Daim duab 4. Inhibition ntawm melanin ntsiab lus (a) thiab cellular tyrosinase kev ua si (b) ntawm BHCP ntawm B16F10 melanoma hlwb.

2.5. Cov teebmeem ntawm BHCP ntawm Kev Tshaj Tawm ntawm MITF / Tyrosinase thiab Phosphorylated CREB hauv B16F10 Cells

MITF, ib qho kev hloov pauv tshwj xeeb, ua lub luag haujlwm tseem ceeb hauv kev ua kom zoo ntawm melanogenicgenes, suav nrog tyrosinase, catalyzing tus nqi txwv cov kauj ruam hauv melanin biosynthesis: TRP-1, thiab TRP-2.Qhov kev qhia ntawm MITF tuaj yeem tau nce los ntawm phosphorylation ntawm CREB [23]. CREB yog ib qho tseem ceeb MITF txhawb nqa [24,25], thiab phosphorylation ntawm CREB hauv melanocytes nce MITF qhia los ntawm kev khi rau CREB (c-AMP teb cov ntsiab lus-binding protein) hauv melanocytes [26]. Txhawm rau elucidatethe molecular pathways lub luag haujlwm rau kev tiv thaiv melanogenic cuam tshuam ntawm BHCP ntawm B16F10 hlwb, peb tau tshuaj xyuas cov protein ntau ntawm cov molecules tseem ceeb, suav nrog CREB thiab MITF, uas ua lub luag haujlwm tseem ceeb hauv melanogenesis los ntawm Western blot tsom xam. Cov hlwb raug kho nrog BHCP lossis kojic acid thiab tom qab ntawd txhawb nqa los ntawm -MSH rau 48 teev. Lub sijhawm luv rau kev ntsuas tau ua raws li cov txheej txheem tau piav qhia hauv cov kev tshawb fawb yav dhau los [27]. Raws li pom hauv daim duab 5, tyrosinase thiab MITF qib nce nrog -MSH, tab sis BHCP txo cov protein ntau. Tsis tas li ntawd, phosphorylation ntawm CREB raug cuam tshuam los ntawm BHCP. Cov kev cai ntawm -MSH-induced CREB phosphorylation paub tias muaj peev xwm tseem ceeb hauv kev tswj cov pigmentation [28]. Cov txiaj ntsig no qhia tau hais tias cov tshuaj tiv thaiv melanogenic ntawm BHCP tshwm sim los ntawm kev txo qis ntawm MITF thiab tyrosinase viathe downregulation ntawm phosphorylated CREB. Txoj kev tshawb fawb tam sim no qhia tau hais tias qhov elucidation ntawm theinhibition mechanism ntawm BHCP ntawm tyrosinase thiab melanogenesis yog qhov tseem ceeb thiab yuav tsum tau tshawb xyuas ntxiv rau yav tom ntej. Txawm hais tias B16F10 melanoma cell kab feem ntau tsim nyog rau kev tshawb nrhiav cov teeb meem hauv vitro, B16F10 cell kab yog ntawm nas melanoma.Yog li ntawd, kev tshawb fawb ntxiv ntawm tib neeg melanocytes yuav tsim nyog los lees paub qhov kev tshawb pom.

2.6. Qhov cuam tshuam ntawm BHCP ntawm UV-Induced NF-κB p-p65 Ua rau hauv Hs27 Cells

Peb tom ntej no tau tshawb xyuas cov txiaj ntsig ntawm BHCP ntawm UV-induced qhia ntawm inflammatory mediatorsusing Hs27 fibroblasts. Nws tau raug tshaj tawm yav dhau los tias phosphorylation ntawm p65 (Ser536) yog qhov tseem ceeb rau nws lub peev xwm los hloov cov noob [29]. Yog li ntawd, cov qib protein ntawm p-p65 (Ser536) thiab p65 tau kuaj xyuas hauv cov nucleus feem los ntawm Western blotting. Raws li tau teev tseg hauv daim duab 6a, b, hauv Hs27cells, UV nce qib protein ntawm p-p65 (Ser536) hauv cov nucleus, qhov kev kho BHCP txo qis cov nucleus p-p65 (Ser536) protein ntau. Ua ke, tag nrho cov nyiaj ntawm p65 tau txo qis hauv cytoplasm los ntawm UV thiab rov qab los ntawm BHCP (Daim duab 6c,d). Txawm hais tias cov protein qhia qib ntawm p65 tau txo qis hauv cytoplasm thiab nce hauv cov nucleus tom qab UV induction, pretreatment nrog BHCP thim cov qauv no nyob rau hauv ib koob tshuaj. Yog li, cov txiaj ntsig no pom zoo tias qhov inhibition ntawm p-p65 los ntawm BHCP tuaj yeem ua rau muaj kev tiv thaiv kev tiv thaiv ntawm daim tawv nqaij thiab kev puas tsuaj collagen tiv thaiv UV.

Daim duab 6. Cov teebmeem ntawm BHCP ntawm kev qhia theem ntawm p-p65 (Ser536) hauv UV-induced Hs27 humanfibroblast cells.

2.7. Qhov cuam tshuam ntawm BHCP ntawm Kev Tshaj Tawm ntawm MMPs hauv Hs27 Cells

MMPs ua lub luag haujlwm tseem ceeb hauv kev laus ntawm daim tawv nqaij. UV irradiation hloov cov ntaub so ntswg ntawm daim tawv nqaij los ntawm upregulating kev qhia ntawm MMPs [30,31]. MMP-1 yog collagen-decomposing enzyme uas ua kom cov collagen synthesized los ntawm hom I procollagen. MMP-9 isa gelatin-decomposing enzyme uas rhuav tshem collagen fibers txiav los ntawm MMP-1, ua rau wrinkleproduction thiab elasticity poob. Txhawm rau tshuaj xyuas cov nyhuv anti-wrinkle ntawm BHCP tiv thaiv UV induction, peb txiav txim siab qib MMP-1 thiab MMP-9 protein los ntawm Western blotting. Tsis tas li ntawd, UV-inducedcells pom tau tias muaj qib siab ntawm MMP-13, uas pib qhov degradation ntawm hom I thiabIII collagen hloov MMP-1 [32]. Peb tau tshawb xyuas qhov nce ntawm MMPs (MMP1, MMP9, MMP12, thiab MMP13) tom qab kho UV-induced Hs27 hlwb nrog BHCP ntawm 1 thiab 10 μM. Raws li pom hauv daim duab 7, qib ntawm MMP-1, MMP-9, MMP-12, thiab MMP-13 qhia tau nce ntxiv tom qab UV induction; txawm li cas los xij, kev kho BHCP txo qis kev qhia hauv ib koob. - kev vam meej. Peb cov txiaj ntsig tau qhia tias BHCP tuaj yeem pab txhawb rau kev tiv thaiv ntawm kev tsim wrinkle los ntawm kev txo qis qhov txawv txav ntawm MMPs vim los ntawm UV raug. Cov txiaj ntsig no txhais tau tias BHCP inhibits MMP qhia hauv Hs27fibroblasts los tiv thaiv collagen decomposition, thiab yog li wrinkle ntau lawm. Yog li, BHCP nthuav tawm lub peev xwm los siv los ua tshuaj tiv thaiv thiab kho cov kab mob ntawm daim tawv nqaij.

cistanche qia cov txiaj ntsig

3. Cov khoom siv thiab cov txheej txheem

3.1. Chemicals thiab Instrumentation

Mushroom tyrosinase (EC 1.14.18.1), -MSH, L-tyrosine, 3, 4-dihydroxyphenylalanine (L-DOPA), dimethyl sulfoxide (DMSO), thiab kojic acid tau muas los ntawm Sigma-Aldrich (St. Louis, MO, USA).Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, streptomycin, and amphotericinwere purchased from Gibco Life Technologies Inc. (Carlsbad, CA, USA). Antibodies tiv thaiv MITF, CREB, p-CREB, p-p65 (Ser536), p65, tyrosinase, MMP-1, MMP-9, MMP-12, MMP-13, TFIIB , thiab -actinwere muas los ntawm Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride (PVDF) membranes tau txais los ntawm Millipore Corporation (Bedford, MA, USA). Sterileplastic ware rau cov ntaub so ntswg kab lis kev cai tau yuav los ntawm SPL Labware (Seoul, Kauslim). UV lightsource yog muab los ntawm Crosslinker 800 series (UVP, CA, USA) 6 lub teeb (8 watts / teeb).thin-txheej chromatography thiab silica gel 60 (mesh 230–400) tau ua ntawm silica gelF{{27} } cov phaj precoated los ntawm Merck Millipore (Darmstadt, Lub teb chaws Yelemees). NMR spectra tau sau tseg siv Varian Unity INOVA 400 (400 MHz rau 1H, 100 MHz rau 13C) thiab Varian Unity AS 500 (500 MHz rau 1H) cov cuab yeej. Chemical shift values ​​(δ) yog qhia nrog rau cov residualsolvent los yog deuterated peaks (δH 2.50 thiab δC 39.51 rau DMSO). Cov ntaub ntawv pov thawj qis-kev daws teeb meem loj spectrometry tau txais nrog Expression CMS huab hwm coj spectrometer (Advion, Ithaca, NY, USA).

3.2. Mushroom Tyrosinase Inhibition Assay

Mushroom tyrosinase inhibitory kev ua haujlwm tau txiav txim siab siv L-tyrosine thiab L-DOPA assubstrates, raws li cov txheej txheem piav qhia los ntawm Jung li al. [23]. Luv luv, 190 μL ntawm tyrosinase enzyme (1000 U diluted nrog nceb tyrosinase tsis, nrog rau 1 mM L-tyrosine thiab L-DOPA tov) tau ntxiv, nyob rau hauv lub xub ntiag los yog tsis muaj cov tebchaw (qhov kawg concentration ntawm 1 mus rau 20 μM, yaj hauv 100 feem pua ​​DMSO), rau txhua qhov dej ntawm 96-paj zoo, muab qhov ntim kawg ntawm 200 μL. Lub phaj yog incubated ntawm 37 ◦C rau 30 min. Tyrosinase kev ua haujlwm tau raug ntsuas los ntawm kev ntsuas qhov nqus ntawm 492 nm siv lub microplate nyeem ntawv (TECAN, Salzburg, Austria) thiab qhov feem pua ​​​​ntawm inhibition (feem pua) tau txais los ntawm cov kab zauv hauv qab no:

feem pua ​​inhibition=(Ac − As)/Ac × 100 (1)

qhov twg Ac yog qhov absorbance ntawm kev tswj thiab As yog qhov absorbance ntawm tus qauv. IC50 qhov tseem ceeb tau suav los ntawm cov log-linear nkhaus thiab lawv qhov sib npaug. Cov txiaj ntsig nruab nrab rau peb qhov kev txiav txim siab tau pom. Kojic acid tau siv los ua kev tswj xyuas zoo.

3.3. Kinetic Analysis ntawm Tyrosinase Inhibition

Txhawm rau txiav txim siab kinetic mechanisms, ob txoj hauv kev kinetic (Lineweaver-Burk thiab Dixon plots) tau siv los ua ke [21,22,33]. Rau Lineweaver-Burk ob chav sib txuas ua ke (nplooj ntawm 1 / enzyme tshaj tawm (1 / V) piv rau 1 / substrate concentration (1 / [S]), hom inhibition tau txiav txim siab siv ntau qhov ntau ntawm L-tyrosine (1, 2, thiab 4 mM) thiab L-DOPA (0.5, 1, thiab 2 mM) raws li substratesin muaj qhov sib txawv ntawm BHCP. Cov concentrations ntawm BHCP yog raws li nram no:0, 0.5, 1.{{20}}, thiab 2.0 μM rau L-tyrosine; thiab 0, 2.5, 5, thiab 10 μM rau L-DOPA. Dixon zaj duab xis yog graphicalmethod (ploj ntawm 1 / enzyme tshaj tawm (1 / V) tawm tsam inhibitor concentration (I)) rau kev txiav txim siab ntawm hom enzyme inhibition thiab tau siv los txiav txim siab qhov sib txawv tsis tu ncua lossis Ki rau theenzyme-inhibitor complex. Dixon cov phiaj xwm (ib lub phiaj xwm sib txuas) ntawm qhov inhibition tau txais thaum muaj L-tyrosine substrate ntawm 1, 2, thiab 4 mM thiab {{40}}, 0.5, 1. {47}}, thiab 2.0 μM rau BHCP; thiab L-DOPAsubstrate ntawm {{50}}.5, 1.0, thiab 2.0 mM thiab 0, 2.5, 5.0, thiab 10.0 μM rau BHCP.

cistanche cogyog tyrosinase inhibitor.

3.4. Cell Kab thiab Cell Culture

Murine B16F10 melanoma hlwb tau txais los ntawm Korean Cell Line Bank. Tib neeg cov tawv nqaij fibroblast cell kab Hs27 tau yuav los ntawm American Type Culture Collection (ATCC, Manassas, VA, USA). Cov hlwb no tau khaws cia hauv DMEM ntxiv nrog 10 feem pua ​​​​fetal bovineserum, 100 U / mL penicillin, thiab 100 mg / mL streptomycin hauv cov av noo 5 feem pua ​​​​CO2 incubatorat 37 ◦C. Dermal fibroblasts ntawm 100 hli phaj tau kho nrog BHCP thiab raug rau 50 mJ / cm2UV hauv DMEM tsis pub dawb (UV lub teeb ci, UVP). Lub Hs27 hlwb tau coj mus rau 70-80 feem pua ​​​​ntawm cov confluencein 100 hli txoj kab uas hla phaj thiab tau siv nyob rau ntawm cov lej 5 thiab 15.

3.5. Cell Viability Assay

Kev muaj peev xwm ntawm cov hlwb raug soj ntsuam siv EZ-Cytox cov khoom ntsuas. Hauv luv luv, B16F10 hlwb thiab Hs27fibroblasts tau muab noob rau hauv 96-cov phaj zoo ntawm qhov ntom ntawm 1 × 104 hlwb / qhov dej thiab incubated ntawm 37 ◦Cfor 24 h. Cov hlwb tau noj tshiab, tsis muaj DMEM uas muaj qhov sib txawv (0, 1, 2, 5, thiab 10 μM) ntawm BHCP, thiab incubated rau 24 thiab 48 teev. Tom qab ntawd, 10 μL ntawm EZ-Cytox tov tau muab tso rau hauv txhua lub qhov dej thiab cov hlwb raug incubated rau 2-4 teev. Kev ntsuas qhov nqus ntawm cov hlwb hauv qhov tsis muaj kev kho mob tau suav tias yog 100 feem pua ​​​​ntawm cov ciaj sia taus. Txhua qhov kev kho mob tau ua tiav intriplicate thiab txhua qhov kev sim tau rov ua peb zaug.

3.6. Kev txiav txim siab ntawm Melanin Contents Assay

Cov nyhuv ntawm BHCP ntawm -MSH-induced melanogenesis nyob rau hauv B16F10 hlwb yog raws li ib tug yav tas los siv txoj kev nrog me ntsis kev hloov [34]. Luv luv, B16F10 hlwb (5 × 104 hlwb/zoo) nyob rau hauv 6-zoo plates raug tso cai kom loj hlob mus rau 70-80 feem pua ​​​​ntawm cov confluence. Tom qab ntawd cov hlwb raug kho nrog qhov sib txawv ntawm BHCP (1, 5, thiab 10 μM) lossis kojic acid (5 mM) rau 24 teev, thiab tom qab ntawd txhawb nqa nrog -MSH (5 μM) rau 48 teev. Tom qab kev kho mob, cov hlwb raug ntxuav ob zaug nrog dej khov-txias PBS, yaj hauv 90 μL 1 M NaOHsolution suav nrog DMSO (5 feem pua) ntawm 60 ◦C rau 1 h, thiab qhov nqus tau ntsuas ntawm 405 nm nrog microplate spectrophotometer (TECAN, Salzburg , Austria). Txhawm rau ntsuas tus nqi melanin hauv kev sim, tus nqi ntawm inhibition hauv pab pawg kho mob tau suav los ntawm kev nqus ntawm cov paub txog cov khoom siv hluavtaws melanin, uas tau raug kho rau tag nrho cov protein uas muaj nyob hauv supernatant ntawm cov cell lysates. Lub absorbance ntawm cov hlwb tsis kho yog ntsuas qhov triplicate.

3.7. Cellular Tyrosinase Activity Assay

Cellular tyrosinase kev ua ub no tau ua los ntawm kev ntsuas tus nqi ntawm oxidation ntawm L-DOPA [35]. B16F10 cov hlwb ntawm qhov ntom ntawm 5 × 104/cells tau muab tso rau hauv 6-cov tais diav zoo thiab incubated thaum hmo ntuj. Tom qab ntawd cov hlwb raug kho nrog ntau qhov ntau ntawm BHCP (1, 5, thiab 10 μM) lossis kojic acid (5 mM) rau 24 teev, thiab tom qab ntawd txhawb nrog -MSH (5 μM) rau 48 teev. Cov hlwb raug ntxuav nrog PBS thiab lysed hauv cov tshuaj uas muaj 100 μL ntawm 50 mM phosphate buffer (pH 6.5), 0.1 mMphenylmethylsulfonylfluoride (PMSF), thiab 1 feem pua ​​​​Triton X-100. Tom qab ntawd, cov hlwb tau muab tso rau hauv lub tshuab nqus dej tob (-80 ◦C) rau 30 feeb. Tom qab defrosting cov hlwb, cov cellular rho tawm tau purified los ntawm centrifugation ntawm 12, 000 rpm rau 30 min ntawm 4 ◦C. Tag nrho ntawm 80 μL lub supernatant thiab 20 μL ntawm L-DOPA (2 mg / mL) tau ntxiv rau hauv 96- lub phaj zoo, thiab qhov nqus ntawm lub wavelength ntawm 492 nm tau ntsuas txhua 10 min hauv 1 h ntawm 37 ◦ C nrog ELISA phaj nyeem ntawv (TECAN, Salzburg, Austria).

3.8. Kev npaj ntawm Cytosolic thiab Nuclear Extracts ntawm Hs27 Cells

Hs27 hlwb raug ntxuav nrog dej khov-txias PBS thiab sau. Ib qho tsis muaj 10 mM Tris(pH 8.0), 1.5 mM MgCl2, 1 mM DTT, 0.1 feem pua ​​​​NP-40, thiab protease inhibitors tau siv rau kev rho tawm ntawm cytosolic feem los ntawm centrifugation ntawm 12, 000 rpm ntawm 4 ◦C rau 15 min, thiab cov seem nuclear raug rho tawm los ntawm cov pellets siv cov tsis muaj 10 mM Tris, 50 mM KCl, 100 mM NaCl, thiab proteaseinhibitors, incubated on ice, thiab tom qab ntawd centrifuged ntawm 13, 000 × g rau 30 min ntawm 4 ◦C kom tau txais cov feem pua ​​​​ntawm cov nuclear.

3.9. Western Blotting

Lysate cov qauv raug muab rhaub rau 10 min hauv gel-loading tsis (125 mM Tris-HCl, 4 feem pua ​​​​sodiumdodecyl sulfate (SDS), 10 feem pua ​​2-mercaptoethanol, thiab 0.2 feem pua ​​​​bromophenol xiav; pH 6.8) ntawm qhov ntim ntawm 1: 1. Tag nrho cov protein sib npaug rau txhua tus qauv tau sib cais los ntawm SDS-polyacrylamide gelelectrophoresis (PAGE) siv acrylamide gels raws li cov txheej txheem piav qhia los ntawm Laemmli [36] thiab xa mus rau PVDF daim nyias nyias ntawm 80 V rau 2 h siv cov txheej txheem ntub dej. Cov daim nyias nyias tau muab tso rau hauv qhov thaiv tsis pub (5 feem pua ​​​​cov mis nyuj tsis muaj rog) hauv 10 mM Tris, pH 7.5, 100 mMNaCl, thiab 0.1 feem pua ​​Tween -20. Cov blots raug thaiv kom tsis txhob muaj kev sib khi ntawm 25 ◦C rau 2 h.Tom qab ntawd, cov daim nyias nyias tau tsim nrog cov tshuaj tiv thaiv tshwj xeeb ntawm 4 ◦C thaum hmo ntuj, tom qab ntawd los ntawm incubation nrog horseradish peroxidase-conjugated secondary antibody ntawm 25 ◦Cfor 1 h. . Antibody labeling tau kuaj pom los ntawm kev txhim kho chemiluminescence raws li tus neeg tsim khoom cov lus qhia. Protein quantitation tau ua los ntawm Davinch-Chemi TM.Chemiluminescence Imaging System CAS-400SM (Core Bio, Seoul, Kauslim). Prestained protein markers tau siv rau kev txiav txim siab ntawm qhov hnyav molecular.

3.10. Kev txheeb cais

Tag nrho cov ntaub ntawv raug nthuav tawm raws li qhov txhais tau tias ± SEM Cov ntaub ntawv tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) rau qhov sib txawv ntawm kev kho mob ua raws li Bonferroni tom qab kev xeem. Tus nqi ntawm p < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">

4. Cov lus xaus

Hauv cov ntsiab lus, qhov kev tshawb pom ntawm txoj kev tshawb fawb tam sim no tau pom tias BHCP muaj cov nyhuv ntawm daim tawv nqaij dawb los ntawm inhibition ntawm tyrosinase, uas yog ib qho tseem ceeb enzyme rau melanin biosynthesis hauv -MSH-induced B16F10melanocytes. Tsis tas li ntawd, BHCP txo qis kev qhia ntawm MMP cov protein ntau hauv UV-inducedfibroblasts, uas xav tias yuav muaj kev tiv thaiv wrinkle. Kev tshawb fawb ntxiv yog yuav tsum tau ua kom paub tseeb tias qhov ua kom tawv nqaij dawb thiab tiv thaiv kab mob ntawm BHCP los ntawm kev tshawb fawb tsiaj thiab kev kho mob. Thaum kawg, BHCP tau txheeb pom tias muaj ob qho tib si ntxuav hniav dawb thiab tiv thaiv kab mob, qhia nws lub peev xwm rau kev txhim kho cov tshuaj kho mob rau cov kab mob cuam tshuam nrog hyperpigmentation thiab wrinkling.

dab tsi yog cistanche

Yog xav paub ntxiv, thov nyem qhov no.