Hauv Vitro Thiab hauv Silico Insights Rau Tyrosinase Inhibitors
Mar 28, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Hee Jin Jung a,b,c, Sang Gyun Noh a,b,c, Yujin Park a, Dongwan Kang a, Pusoon Chun d, Hae Young Chung a,b,c,⇑,Hyung Ryong Moon a
Abstract
Tyrosinase yog ib qho tseem ceeb enzyme lub luag hauj lwm rau melanin biosynthesis thiab zoo tiv thaiv daim tawv nqaij puas los ntawm ultraviolet hluav taws xob. Raws li ib feem ntawm kev siv zog txuas ntxiv mus nrhiav cov muaj zog tyrosinase inhibitors, wesystematically tsim thiab synthesized kaum peb (E)-benzylidene-1-indanone derivatives (BID1-13) thiab txiav txim siab lawv cov haujlwm inhibitory tiv thaiv tyrosinase. Ntawm cov tshuaj ntsuam xyuas, BID3 yog qhov muaj zog tshaj plaws inhibitor ntawm nceb tyrosinase (IC50=0.034 mM, mycophenolate kev ua haujlwm; IC50=1.39 mM, diphenols kev ua haujlwm). Kinetic cov kev tshawb fawb tau qhia tias BID3 tau pom ib hom kev sib xyaw ntawm tyrosinase inhibition nrog Ki tus nqi ntawm 2.4 mM siv L-DOPA ua substrate. Hauv silico molecular docking simulations pom tau hais tias BID3 tuaj yeem khi rau qhov chaw catalytic thiab allosteric ntawm tyrosinase los inhibit enzyme kev ua haujlwm uas tau lees paub hauv vitro kev tshawb fawb ntawm BID3 thiab tyrosinase. Tsis tas li ntawd, cov ntsiab lus melanin txo qis thiab kev ua haujlwm ntawm cellular tyrosinase inhibited tom qab kev kho BID3. Cov kev soj ntsuam no tau nthuav tawm tias BID3 yog ib qho muaj zog tyrosinase inhibitor thiab muaj peev xwm siv tau los ua cov tshuaj pleev xim dawb rau kev kho mob ntawm cov xim pigmentation.
Cistanche: daim tawv nqaij whitening tshuaj ntsuab
1. Taw qhia
Melanin yog synthesized los ntawm melanocytes nyob rau hauv vassal txheej ntawm theepidermis feem ntau yog hais txog tsev neeg ntawm cov pigments feem ntau paub txog kev tiv thaiv mammalian daim tawv nqaij los ntawm kev puas tsuaj los ntawm kev puas tsuaj los ntawm UV hluav taws xob los ntawm scavenging dawb radicals los yog dispersingincoming UV teeb [1]. Txawm li cas los xij, ntau tiam ntawm melanincan ua rau pom pom hyperpigmentation ntawm epidermis, uas tej zaum yuav pom tseeb xws li melasma, freckles, hnub nyoog me ntsis, los yog senile lentigines [2].
Tyrosinase (EC 1.14.18.1), tooj liab-muaj metalloenzyme, yog ib qho tseem ceeb enzyme koom nrog hauv cov txheej txheem melanogenic. Tyrosinase catalyzes ob theem-txheej txheem hauv melanogenesis; hydroxylation ntawm tyrosine (cresolase los yog mycophenolate kev ua si) los tsim 3, 4-dihydroxyphenylalanine (L-DOPA) thiab tom qab oxidation ntawm L-DOPA (catecholase los yog diphenols kev ua si) rau qhov sib thooj DOPA-quinone. Thaum L-tyrosine yog lub substrate, cov khoom ntawm tyrosinase catalyzed cov tshuaj tiv thaiv yog DOPA-quinone, uas yog ces hloov mus rau melanin [3]. Cov kauj ruam no yog qhov tseem ceeb rau kev tiv thaiv ntawm daim tawv nqaij tiv thaiv UV kev puas tsuaj. Mushroom Agaricus yog siv rau nws txoj kev lag luam muaj thiab siab homology nrog mammaliantyrosinase enzyme uas ua rau nws zoo haum raws li tus qauv rau kev kawm txog melanogenesis [4]. Hauv tib neeg, melanin pab tiv thaiv daim tawv nqaij los ntawm kev puas tsuaj los ntawm UV [5]. Txawm li cas los xij, ntau dhau ntawm melanincan ua rau ntau yam kab mob dermatological xws li hyperpigmentations, melasma, freckles, thiab hnub nyoog me ntsis [6]. Yog li ntawd, newtyrosinase inhibitors pom cov tshuaj zoo li thiab daim tawv nqaij-whiteningproperties yuav tsum tau inhibiting ntau ntawm daim tawv nqaij pigmentation.
1-Indanone, nrog lub cev pob txha benzoyl-cyclopentanone, raug suav hais tias yog cov kwv tij txheeb ze ntawm chalcones, suav nrog cov ketone tsis txaus ntseeg ntawm chalcones tsim lub voj voog 5 lub nplhaib, uas yog ib qho tshwm sim ib txwm muaj tshwm sim tam sim no invarious edible cog qhov chaw [7 ]. Ntau qhov kev tshawb fawb tau pom tias muaj cov khoom sib xyaw ua ke muaj 1-indanone moiety muaj cov tshuaj tseem ceeb, raws li lawv nthuav tawm ntau yam kev ua haujlwm lom neeg, suav nrog tshuaj tiv thaiv kab mob [8–10], tshuaj tiv thaiv kab mob [11,12], antioxidant [13], tiv thaiv Parkinson's kab mob [14], anti-Alzheimer kab mob [15–17], tshuaj tua kab mob [18], tiv thaiv kab mob tiv thaiv kab mob [19], andanti-tyrosinase [20] zog.
Chalcones (1,3-diaryl-2-tsim-1-ones) yog cov muaj ntxhiab ketones uas muaj ob lub nplhaib uas muaj ntxhiab txuas nrog peb-carbon a, b-unsaturated carbonyl system ua lub hauv paus tseem ceeb [21, 22] ib. Cov khoom no tshwm sim nyob rau hauv ntau yam edible naturalplants thiab xam raws li precursor tebchaw rau ib tug synthetic routeof flavonoids thiab isoflavonoids xws li pyrazolines, pyrimidine, flavanol, flavones, flavanones, isoflavones, aurones, anthocyanidin, dihydroflavanol 3-2. Lub a, b unsaturated carbonyl system yog qhov tseem ceeb rau hom kev ua haujlwm lom neeg, txij li cov tshuab no tau muab faib rau hauv ntau yam khoom ntuj xws li chalcones thiab indanone, qhov twg cov molecular yog cov qauv ntau dua, thiab kev sib kis ntawm hluav taws xob cuam tshuam ntawm aryl substituents yog qhia. rau carbonyl pawg [7] Yav dhau los ntau tus kws tshawb fawb tau tshaj tawm tias chalcones tau qhia tias yog cov chav kawm tshiab ntawm tyrosinase inhibitor hauv ntau qhov kev tshaj tawm [26–29]. Tsis ntev los no, Kim thiab cov neeg ua haujlwm sib koom ua ke [30,31] tsim thiab tsim cov aseries ntawm chalcone derivatives thiab ntsuas lawv rau lawv cov tshuaj tiv thaiv vitro. tyrosinase thiab anti-melanogenic kev ua ub no tiv thaiv murine melanocytes.
Raws li peb cov ntaub ntawv yav dhau los qhia, derivatives nrog ib (E)-b-phenyl-a, b-unsaturated carbonyl scaffold pom potenttyrosinase inhibition [32-34]. (E)-Benzylidene-1-indanones tuaj yeem ua tus neeg sawv cev zoo nkauj los tiv thaiv melanogenic txij li cov compoundshave (E)-b-phenyl-a, b-unsaturated carbonyl scaffold hauv lawv cov qauv tshuaj. Tshwj xeeb, Radhakrishnan et al. (2015) [20] tsim thiab sib txuas ua ke ntawm benzylidene-1-indanonederivatives bearing a (Z)-configuration thiab ntsuam xyuas lawv foranti-tyrosinase kev ua. Txawm hais tias cov (Z)-benzylidene-1-indanone derivatives tau tshawb xyuas anti-tyrosinase thiab anti-melanogenic cov dej num ntawm (E)-benzylidene-1-indanone derivatives tseem tsis tau raug tshuaj xyuas.
Yog li ntawd, peb tsim thiab ua ke kaum peb lub tebchaw (BID1–13) ntawm 1-indanone skeleton bearing ib (E)-form benzylidene. Ntawm cov khoom siv hluavtaws no, ib qho 2, 4-dihydroxygroup ntawm lub nplhaib B ntawm 1-indanone pom qhov zoo tshaj plaws tyrosinaseinhibition (kwv yees 400-fold zoo tshaj kojic acid, kev tswj zoo). Ntxiv mus, peb ntxiv kev soj ntsuam BID3 tyrosinaseinhibitory kev ua ub no nrog rau kev ua kom nws lub luag haujlwm tswj hwm hauv melanin synthesis siv B16F10 melanoma hlwb. Hauv kev sim txuas ntxiv, peb tau soj ntsuam qhov muaj peev xwm tiv thaiv melanogenic ntawm BID3 thiab nrhiav kev txheeb xyuas cov enzyme kinetics thiab molecular dockingstudies. Hauv kev sim ua tiav, BID3 cellular tyrosinase muaj peev xwm thiab melanin ntau lawm hauv a-MSH thiab IBMX-induced B16F10 melanoma hlwb kuj tau tshawb nrhiav.
Anti-Oxidation thiab tawv nqaij nrog rau: CISTANCHE EXTARCT
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Reagents
Mushroom tyrosinase (EC 1.14.18.1), alpha-melanocyte-stimulating hormone (a-MSH), 3-isobutyl-1-methylxanthine (IBMX), L-tyrosine, L-3, {{ 11}}dihydroxyphenylalanine (L-DOPA), dimethyl sulfoxide (DMSO), kojic acid, phthalic acid, thiab trans-cinnamic acid tau yuav los ntawm Sigma-Aldrich (St. Louis, MO, USA). Dulbecco'smodified Eagle's medium (DMEM), fetal bovine serum (FBS), streptomycin, thiab amphotericin tau yuav los ntawm WELGENE Inc.(Gyeongsan-si, South Kauslim). Tag nrho lwm cov reagents tau yuav los ntawm Sigma-Aldrich.
2.2. Chemistry
2.2.1. Cov txheej txheem dav dav
Tag nrho cov reagents tau txais kev lag luam thiab siv yam tsis muaj kev lim dej ntxiv. Nyias-txheej chromatography (TLC) thiab columnchromatography tau ua nyob rau Merck precoated 60F245plates thiab MP Silica 40–63, 60 Å, feem. Cov ntaub ntawv daws teeb meem siab (HR) huab hwm coj spectroscopy tau txais ntawm Agilent AccurateMass quadruple-time of flight (Q-TOF) kua chromatography (LC) mass spectrometer (Agilent, Santa Clara, CA, USA) hauv ESI positivemode. Nuclear magnetic resonance (NMR) spectra tau sau rau ntawm Varian Unity INOVA 400 spectrometer los yog Varian UnityAS500 spectrometer (Agilent Technologies, Santa Clara, CA, USA) rau 1H NMR (400 thiab 500 MHz) thiab rau 13C NMR (100 MHz), ntsig txog. . DMSO d6, CD3OD, thiab CDCl3 tau siv los ua NMR cov kuab tshuaj rau NMR cov qauv. Coupling tas li (J) thiab tshuaj hloov pauv tau ntsuas hauv hertz (Hz) thiab qhov chaw ib lab (ppm), feem. Cov ntawv luv uas siv rau hauv kev tshuaj xyuas ntawm 1H NMR data yog raws li hauv qab no: s (singlet), bs (broad singlet), d (ob chav), dd (ob npaug ntawm doublets), t (triplet), TD (triplet ntawm doublets), q ( quartet), andm (ntau).
2.2.2. Cov txheej txheem rau kev sib txuas ntawm BID1-13
Ib qho kev daws ntawm benzaldehyde (1.1–1.2 Equiv.) thiab 1-indanone (100 mg, 0.76 mmol) hauv 1 M hauv HCl acetic acid (0.4 mL) yog stirredat chav tsev kub. rau 4-48 h. Tom qab ntxiv dej, cov precipitates tau lim thiab ntxuav nrog dej thiab hexane: ethylacetate (1: 1), hexane: dichloromethane (4: 1-1: 1), los yog sib tov ntawm dichloromethane thiab methanol, nyob ntawm seb cov khoom ntawm cov benzaldehydes seem. muab (E)-benzylidene-1-indanones (BID1–13) hauv yields ntawm 32.8–99.1 feem pua. Tus yam ntxwv ntawm tus yam ntxwv (1H thiab 13C NMR thiab cov ntaub ntawv loj ntawm tag nrho cov BIDs) ntawm cov khoom sib txuas tau muab rau hauv cov ntaub ntawv ntxiv.
2.2.2.1. (E)-2-(4-Hydroxybenzylidene)-2,3-dihydro-1H-inden-1-ib (BID1).
Yellow khoom; yog ', 93,4%; lub sijhawm tiv thaiv, 6 h; molecularformula, C16H12O2; mp, 224.7–225.9 C; 1H NMR (400 MHz, DMSO d6) d 10.10 (s, 1H, OH), 7.72 (d, 1H, J=7.6 Hz, 7-} H), 7.64–7.58 (m, 4H, 4-H, 5-H, 20-H, 60-H), 7.43 (s, 1H, vinylic H), 7.39 (t, 1H, J=7.2 Hz, 6-H), 6.87 (d, 2H, J=8.0 Hz, 30-H, {{46 }}H), 3.99 (s, 2H, CH2); 13C NMR (100 MHz, DMSO d6) d 193.9, 160.1, 150.4, 138.3,135.1, 134.0, 133.6, 132.2, 128.2, 127.2, MS1212. H) ntxiv calcd 237.0910, obsd 237.0911.
2.2.2.2. (E)-2-(3,4-Dihydroxybenzylidene)-2,3-dihydro-1H-inden-1-ib (BID2).
Yellow khoom; yield, {{0}}.6 feem pua ; lub sijhawm tiv thaiv, 5 h; Cov mis mos molecules, C16H12O3; mp, 251.2–252.4 C; 1H NMR (400 MHz, DMSO d6) d 9.65 (s, 1H, OH), 9.24 (s, 1H, OH), 7.72 (d, 1H, J=7} .6 Hz, 7-H), 7.66–7.61 (m, 2H, {{30}}H, 5-H), 7.42 (t, 1H, J {{35} }}.2 Hz, 6-H), 7.35 (s, 1H, vinylic H), 7.19 (s, 1H, 20-H), 7.08 (d,1H , J=8.0 Hz, 60-H), 6.83 (d, 1H, J=8.0 Hz, 50-H), 3.98 (s, 2H, CH2 ); 13C NMR (100 MHz, DMSO d6) d 193.8, 150.4, 148.7, 146.3,138.3, 135.1, 134.5, 132.0, 128.2, 127.3, 121. HRMS (ESI plus ) m/z C16H13O3 (M plus H) plus calcd253.0859, obsd 253.0857.
2.2.2.3. (E)-2-(2,4-Dihydroxybenzylidene)-2,3-dihydro-1H-inden-1-ib (BID3).
Yellow khoom; yield, 5{{20}}.9 feem pua ; cov tshuaj tiv thaiv lub sij hawm, 10 h; Cov mis mos molecular, C16H12O3; mp, 198.5–199.9 C (dec.); 1H NMR (500 MHz, CD3OD) d 8.17 (s, 1H, vinylic H), 7.82 (d, 1H, J=8.0 Hz,60-H), 7.67–7.63 (m, 3H , 4-H, 5-H, 7-H), 7.45 (t, 1H, J=7.0 Hz, 6-H), 6.45 (d , 1H, J=8.5 Hz, 50-H), 6.39 (s, 1H, 30-H), 4.01 (s, 2H, CH2); 13C NMR (100 MHz, DMSO d6) d 193.9, 161.6, 160.4, 150.3,138.6, 134.8, 131.7, 130.3, 128.7, 128.1, 120.3, 112. HRMS (ESI plus ) m/z C16H13O3 (M plus H) plus calcd253.0859, obsd 253.0858.
2.2.2.4 ib. (E)-2-(4-Hydroxy-3-methoxybenzylidene)-2, 3-dihydro-1Hinden-1-ib (BID4).
Yellow khoom; yield, 52.0 feem pua ; cov tshuaj tiv thaiv lub sij hawm, 4 h; molecular mis, C17H14O3; mp, 186.9–187.4 C; 1H NMR(400 MHz, DMSO d6) d 9.71 (s, 1H, OH), 7.73 (d, 1H, J=7}.6 Hz, 7- H), 7.67–7.62 (m, 2H, 4-H, 5-H), 7.45 (d, 1H, J=2).{{50}} Hz , 20-H), 7.43(t, 1H, J=7.2 Hz, 6-H), 7.31 (s, 1H, vinylic H), 7.24 (dd, 1H, J=8.0,2.0 Hz, 60-H), 6.87 (d, 1H, J=8.0 Hz, 50-H), 4.05 (s, 2H, CH2 ), 3.84(s, 3H, OCH3); 13C NMR (100 MHz, DMSO d6) d 193.9, 150.5,149.7, 148.5, 138.3, 135.2, 134.4, 132.3, 128.2, 127.3, 125.5, 127.5, 127.5, 127., 127. HRMS (ESI plus ) m/z C17H15O3(M plus H) plus calcd 267.1016, obsd 267.1016.
2.3. Kev ntsuam xyuas kev lom neeg
2.3.1. Mushroom tyrosinase inhibitory assay
Tyrosinase inhibitory kev ua ub no tau ua tiav siv cov nceb tyrosinase raws li tau piav qhia yav dhau los, nrog kev hloov pauv me me [35,36]. Luv luv, 10 mL ntawm ib qho kev qhia tshwj xeeb ntawm txhua qhov sib xyaw (0.0005-50 mM) thiab 20 mL ntawm nceb tyrosinase (1000 units / mL) hauv 50 mM phosphate buffer (pH 6.5) tau ntxiv rau 170 mL cov tshuaj tiv thaiv. sib tov nyob rau hauv ib tug 96-zoo microplate (Corning, USA).Qhov piv ntawm 1 mM L-tyrosine los yog L-DOPA tov, 50 mM potassiumphosphate buffer (pH 6.5), thiab distilled dej yog 10:10:9. Cov tshuaj sib tov tau muab tso rau ntawm 37 C rau 30 feeb. Tom qab ntawd, tus nqi ntawm dopachrome tsim nyob rau hauv txhua lub qhov dej yog ntsuas los ntawm spectrophotometric tsom ntawm 492 nm (OD492) siv lub microplate nyeem ntawv. Tus nqi tyrosinase inhibition (feem pua) yog xam raws li (1 Abssample / Abscontrol) 100 feem pua. Cov qib ntawm cov qauv inhibition tau qhia raws li qhov xav tau ntawm 50 feem pua inhibition (IC50).
2.3.2. Enzyme kinetic tsom xam nrog tyrosinase
Txhawm rau txiav txim siab lub kinetic mechanisms ntawm inhibition, ob txoj kev kinetics ntxiv tau siv: Lineweaver-Burk thiab Dixonplots [37–39]. Siv Lineweaver-Burk plots (ob chav reciprocalplots), hom inhibition ntawm BID3 tau txiav txim siab siv ntau yam concentrations ntawm L-DOPA (0.125, 0.25, 0.5, thiab 1. mM), raws li cov substrates, thaum tsis muaj thiab muaj ntau qhov ntau ntawm BID3 (1, 2, thiab 4 lM). Ib daim phiaj Dixon (ib lub phiaj xwm sib koom ua ke) fortyrosinase inhibition tau txais nyob rau hauv muaj ntau qhov ntau ntawm L-DOPA ({17}}.125, 0.25, 0.5, thiab 1 mM ). Qhov siab ntawm BID3 yog 1, 2, thiab 4 mM. Cov txheej txheem enzymatic uas tau piav qhia yav dhau los tyrosinase txoj kev kuaj mob.Qhov inhibition tas li (Ki) tau txiav txim siab los ntawm kev txhais lus ntawm Dixon plots.
2.3.3. Tyrosinase molecular docking simulations
Txhawm rau txiav txim siab tus qauv ntawm cov enzyme-inhibitor complexand kom paub tseeb tias qhov tseeb, rov ua dua, thiab kev ntseeg siab ntawm cov txiaj ntsig docking, peb ua haujlwm AutoDock4.2 software. Rau cov kev tshawb fawb dicking, cov qauv siv lead ua ntawm cov nceb tyrosinase protein lub hom phiaj tau txais los ntawm cov protein sib txuas ua ke (Protein Data Bank (PDB ID: 2Y9X) (http://www.rcsb.org/adb) [40]. Automated docking simulations tau ua. nruab nrab ntawm tyrosinase thiab kojic acid, phthalic acid, cinnamic acid, los yog BID3. Rau cov txheej txheem docking: hloov 2D rau hauv 3D qauv, suav nqi, thiab ntxiv hydrogen atoms siv ChemOffice program (http://www.cam bridgesoft.com) [41 LigandScout 4.1.5 tau siv rau kev kwv yees txog kev sib cuam tshuam ntawm ligands thiab tyrosinase thiab kev txheeb xyuas cov tshuaj pharmacophores.
2.3.4. Cell culture thiab cell viability assay
Murine melanoma B16F10 hlwb tau yuav los ntawm Kauslim Cell Line Bank (KCLB, Seoul, Kauslim Teb) thiab kab lis kev cai hauv DMEM ntxiv nrog 10 feem pua FBS thiab 1 feem pua streptomycin, thiab tom qab ntawd incubatedat 37 C, humidified nrog 5 feem pua atmospheric CO2. Cell viability analyses tau ua raws li tau piav qhia yav dhau los [42]. Luv luv, cov noob qoob loo ntawm qhov ntom ntawm 1 104 hlwb/zoo hauv 96-zoo phaj rau 24 teev. Hnub tom qab, cov hlwb raug nthuav tawm rau ntau qhov sib txawv ntawm BID3 thiab incubated rau 24 los yog 48 h, raws li, 10 mL EZ-Cytox tov tau ntxiv rau txhua lub qhov dej thiab cov cell raug incubated rau 2-4 h. Absorbance tau txiav txim siab siv ELISAat lub wavelength ntawm 450 nm. Txhua qhov kev ntsuam xyuas tau ua hauv triplicate.
2.3.5. Kev ntsuam xyuas melanin
Cov ntsiab lus melanin tau txiav txim siab raws li tau piav qhia yav dhau los [43].B16F10 melanoma hlwb (2 105 hlwb/zoo) tau muab noob rau hauv 6-cov kab lis kev cai zoo. Txhawm rau txiav txim siab cov nyhuv inhibitory ntawm BID3 onmelanogenesis, qhov nruab nrab tshiab tau hloov nrog qhov nruab nrab muaj ing BID3 (1, 5, thiab 10 lM) lossis kojic acid (10 mM) raws li kev tswj hwm zoo rau 1hr, thiab tom qab ntawd txhawb nqa nrog a-MSH (5 lM). ) thiab IBMX (200 mM) rau 48 h. Tom qab ntxuav ob zaug nrog PBS, cov hlwb raug tshem tawm los ntawm kev tsim tawm hauv Trypsin / EDTA, thiab cov pellets tau yaj hauv 100 mL ntawm 1 N NaOH, thiab tom qab ntawd incubated ntawm 60 C rau 1 tes sib xyaw kom solubilize melanin. Cov ntsiab lus melanin tau txiav txim siab los ntawm kev ntsuas qhov nqus ntawm 405 nm los ntawm kev siv ELISA nyeem ntawv (TECAN, Sunrise, Austria). Cov ntsiab lus melanin raug suav nrog kev sib npaug hauv qab no: (cov qauv / tswj) - 100 feem pua .Txhua qhov kev txiav txim siab tau ua hauv triplicate.
2.3.6. Kev ua haujlwm ntawm cellular tyrosinase
Cellular tyrosinase kev ua haujlwm tau raug soj ntsuam raws li tau piav qhia yav dhau los, nrog kev hloov kho me ntsis [44,45]. Luv luv, 2 105/cells tau muab tso rau hauv 6-cov tais diav thiab incubated thaum hmo ntuj. Cov hlwb raug nthuav tawm rau ntau qhov sib txawv ntawm BID3 (1, 5, thiab 10 mM) lossis kojic acid (10 mM) rau 1 h, thiab tom qab ntawd txhawb nqa nrog a-MSH (5 mM) thiab IBMX (200 mM) rau 48 h. Tom qab tau yaug ob zaug nrog PBS, cov hlwb tau lysed nrog 100 mL lysis tov uas muaj 50 mM phosphate tsis (pH 6.5), 0.1 mM phenylmethylsulfonylfluoride (PMSF), thiab 1 feem pua Triton X-100 thiab khov ntawm 80 min. Lysates tau thawed thiab centrifuged ntawm 12, 000 rpm rau 30 min ntawm 4 C. Tom qab ntawd, supernatant (80 mL) tau muab sib xyaw nrog 2 mg / mL L-DOPA (20 mL) hauv 96- zoo phaj . Tom qab incubationat 37 C rau 30 min, qhov optical ceev ntawm 492 nm yog ntsuas los ntawm ELISA nyeem ntawv (TECAN, Salzburg, Austria). Cov protein concentration tau txiav txim siab siv BCA protein kuaj reagent siv BCA raws li tus qauv (Thermo Scientific, Rockford, IL, USA).
Cistanche extract muaj txiaj ntsig: inhibits tyrosinase.
2.3.7 ib. Kev txheeb cais
Tag nrho cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± tus qauv yuam kev ntawm qhov nruab nrab (SEM). Cov ntaub ntawv tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) los txiav txim qhov sib txawv ntawm kev kho mob, ua raws li Bonferroni posthoc test. Tus nqi ntawm p < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">
3. Cov txiaj ntsig thiab kev sib tham
3.1. Chemistry
Cov (E)-benzylidene-1-indanones derivatives (BID1–13) tau ua ke raws li cov txheej txheem dav dav 1. Hauv qhov kev tshawb fawb no, kaum peb (E)-2-benzylidene-1-indanone derivatives tau ua ke thiab lawv tyrosinase inhibitory zog tau kuaj xyuas. Lub hom phiaj (E)-2-benzylidene-1-indanone derivatives tau tsim los ua haujlwm acidic (1 M HCl hauv acetic acid) cov xwm txheej.Cov tshuaj tiv thaiv no tsim lub hom phiaj (E)-2-benzylidene-1- indanone derivatives nyob rau hauv yield ntawm 32.8-99.1 feem pua. Nws zoo nkaus li tias tsuas yog E-isomers ntawm 2-benzylidene-1-indanones raug tsim los ntawm A-strain hu ua 1,{21}}allylic strain. A-strain (sterichindrance) nruab nrab ntawm lub nplhaib phenyl thiab carbonyl pawg hauv Z-isomer yog pom tau tias loj dua li ntawm phenyl ring thiab pawg methylene hauv E-isomer. Cov qauv ntawm cov khoom sib txuas tau raug txheeb xyuas los ntawm 1H thiab 13C NMR thiab huab hwm coj spectrometry raws li tau piav qhia hauv ntu kev sim (Figs. S1-S38).Tshwj xeeb, kev hloov tshuaj lom neeg ntawm olefinic proton yog thaiv hauv E-isomer ntawm 2- benzylidene vim cov nyhuv anisotropic ntawm pawg carbonyl thiab tshwm sim downfield (saum toj no 7 ppm) piv rau Z isomer (<7 ppm)="" [46].="" the="" chemical="" shifts="" of="" olefinic="" protons="" in="" the="" benzylidene-1-indanones="" appeared="" in="" the="" range="" of="" 7.31–8.17="" ppm.="" therefore,="" the="" benzylidene-1-indanone="" derivatives="" were="" determined="" to="" be="" (e)-stereoisomers.="" we="" found="" that="" the="" presence="" of="" hydroxyl="" or="" methoxyl="" group="" at="" the="" 2-position="" of="" the="" phenyl="" ring="" moves="" the="" chemical="" shift="" of="" the="" olefinic="" proton="" to="" 0.5–0.8="" ppm="">
Scheme 1. Synthesis of (E)-2-benzylidene-1-indanones.
3.2. Tyrosinase inhibitory zog ntawm cov tebchaw
Lub peev xwm tyrosinase inhibitory ntawm (E)-benzylidene-1-indanones derivatives tau tshuaj xyuas siv nceb tyrosinase.The mycophenolate (L-tyrosine) thiab diphenols (L-DOPA) tau siv los ua cov substrates rau cov kev sim no [35]. Enzyme cov tshuaj tiv thaiv tau ua nrog tyrosinase, substrate, thiab kuaj inhibitors.Tag nrho cov tshuaj ntsuam xyuas tau pom tias muaj qhov concentration-dependentinhibition. IC50 qhov tseem ceeb ntawm (E)-benzylidene-1-indanonesderivatives tau qhia hauv Table 1.
Hauv ob qho tib si L-tyrosine thiab L-DOPA substrates, BID3 tau nthuav tawm cov haujlwm muaj zog tiv thaiv nrog IC50 qhov tseem ceeb ntawm 0.{{10}}34 ± 0.{{ 27}}224 mM thiab 1.39 ± 0.00004 mM, ntsig txog, piv nrog cov tswj zoo kojic acid, uas muaj IC50 tus nqi ntawm 13.77 ± 0.20 mM thiab 33.14 ± 0.93 mM, feem. (Fig. 1A). Tsis tas li ntawd, BID6 tau qhia tias muaj zog ua haujlwm rau L-tyrosine thiab L-DOPA nrog IC50 qhov tseem ceeb ntawm 5.00 ± 0.91 mM thiab 53.11 ± 2.79 mM, feem. BID1 tau pom tias muaj kev cuam tshuam tsis zoo rau tyrosinase ntawm L-tyrosine thiab L-DOPApathways, nrog rau IC50 qhov tseem ceeb ntawm 39.74 ± 3.71 mM thiab 130.0 ± 5.39 mM, feem. BID4 thiab BID11 tso tawm moderatetyrosinase inhibitory kev ua haujlwm. Lwm cov derivatives tsis ua haujlwm. Ib yam li ntawd, BID2 tau pom cov haujlwm tseem ceeb rau L-tyrosine thiab L-DOPA, nrog rau IC50 qhov tseem ceeb ntawm 41.27 ± 3.03 mM thiab 52.93 ± 2.62 mM. Tsis tas li ntawd, tau tshaj tawm cov tshuaj sib xyaw ua ke, phthalic acid thiab cinnamic acid tsis muaj inhibitory. cov nyhuv ntawm qhov concentration ntawm 200 mm [47].
Raws li qhov kev tshawb pom ntawm cov qauv-kev sib raug zoo kev sib raug zoo (SAR) kev tshawb fawb, derivatives uas muaj (E)-benzylidene-1-indanonesubstituents ntawm lub nplhaib phenyl noticeably cuam tshuam lub peev xwm tyrosinase inhibition. BID3, uas inhibited tyrosinase nrog lub zog siab tshaj plaws, dais ib pab pawg 2-hydroxy nyob rau hauv lub xub ntiag ntawm ib pawg 4-hydroxy thiab inhibited heev tyrosinase kev ua (BID1 vsBID3). Cov txiaj ntsig no tau qhia tias 2, 4- pawg dihydroxy (resorcinol moiety) hauv cov qauv phenyl nplhaib tuaj yeem ua lub luag haujlwm rau kev txhim kho inhibition ntawm tyrosinase. Interestingly, nyob rau hauv ib tug yav dhau los kev tshawb fawb ntawm tej zaum synthetic tyrosinase inhibitors, peb tau pom tias 2,{11}}dihydroxy hloov potently inhibited tyrosinase kev ua si [48,49]. Tsis tas li ntawd, peb cov ntaub ntawv SAR kuj tau qhia tias qhov kev taw qhia ntawm 3- pab pawg hydroxy nyob rau hauv lub xub ntiag ntawm 4-hydroxygroup cuam tshuam rau tyrosinase inhibition. Cov xwm txheej no tau tshwm sim los ntawm kev tshawb pom tias ib qho 4-methoxy-3-hydroxy phenyl ringincreased inhibitory kev ua ub no tiv thaiv tyrosinase, whereas kev taw qhia ntawm ib tug 3, 4-dihydroxy pawg (catechol moiety) txo tyrosinase inhibitory kev ua ( BID2 vs BID6) [35]. Cov txiaj ntsig no qhia tias 3-hydroxy-4- pawg methoxy kuj yog lub luag haujlwm rau kev ua haujlwm fortyrosinase inhibitory. Txawm li cas los xij, hauv peb txoj kev tshawb fawb tam sim no, BID9 thiab BID11, uas muaj 2, 4-dimethoxy phenyl lossis 3, 5-dimethoxy phenyl pawg, tau pom tias muaj tyrosinase inhibitoryactivity. Raws li IC50 qhov tseem ceeb ntawm L-tyrosine thiab L-DOPA txoj hauv kev, BID3 tau pom tias muaj zog tshaj plaws tyrosinase inhibitoryactivity yog li ntawd, peb tau kawm ntxiv txog cov txheej txheem hauv qab no inhibitory nyhuv. Radhakrishnan et al. (2015) [20] qhia tias hydroxyl hloov (Z)-benzylidene-1-indanone compoundspotent tyrosinase inhibitors. Txawm hais tias yav dhau los cov kev tshawb fawb tau pom tias hydroxyl-substituted (Z)-benzylidene-1-indanone derivativespossess anti-tyrosinase kev ua, rau qhov zoo tshaj plaws ntawm peb txoj kev paub, tsis tau muaj lus ceeb toom ntawm tyrosinase inhibition los ntawm (E)-benzylidene-1- indanone derivatives siv cov kev sim ntawm tes.
Table 1. Mushroom tyrosinase inhibitory peev xwm ntawm qhov hloov pauv 2, 3-dihydro-1H-inden-1-ib qho (1-indanone) chalcone-zoo li
derivatives (BID1–13).
Daim duab 1. Concentration-dependent inhibition ntawm nceb tyrosinase kev ua los ntawm BID3 thiab kojic acid (zoo tswj) (n=3).
3.3. Enzyme kinetic tsom xam ntawm tyrosinase inhibition
Txij li thaum BID3 yog tus muaj zog tshaj inhibitor, peb tau kawm ntxiv txog cov txheej txheem hauv qab nws cov nyhuv inhibitory. Hauv kev sim piav qhia txog hom enzyme inhibition, kinetic tsom xam tau ua nyob rau ntawm ntau yam L-DOPA thiab ntau BID3 concentrations. Cov phiaj xwm Dixon thiab Lineweaver-Burk tau kos siv cov ntaub ntawv tau los ntawm cov kev tshawb fawb kinetic kom paub meej tias cov qauv inhibition thiab qhov inhibition tas li (Ki) tau txiav txim siab los ntawm kev txhais lus ntawm Dixon plots (Fig. 2A thiab B). Qhov concentration ntawm L-DOPA yog qhia raws li nram no: 1, 2, 4 mM. Qhov kev sib tshuam nyob rau sab laug, qhia txog kev sib xyaw ua ke tiv thaiv tyrosinase nrog tus nqi Ki ntawm 2.4 mM (Fig. 2B). Tus nqi Ki sawv cev rau qhov kev xav tau los tsim cov enzyme-inhibitorcomplex, yog li tus inhibitor nrog tus nqi qis dua qhia tias muaj ntau dua tyrosinase inhibition.
Molecular docking tau ua rau muaj kev nkag siab tseem ceeb hauv kev tshawb nrhiav tshuaj hauv ntau xyoo. Txawm li cas los xij, cov txheej txheem docking taw qhia qhov tseeb txoj haujlwm ntawm ligands nyob rau hauv lub hnab ntawv khi ntawm aprotein thiab kwv yees qhov kev sib raug zoo ntawm ligand thiab protein. Hauv lwm lo lus, docking piav qhia txog txheej txheem los ntawm ob lub molecules haum ua ke hauv qhov chaw peb sab. Txhawm rau txiav txim siab seb BID3 khi rau qhov chaw nquag ntawm tyrosinase, kev tshuaj xyuas moleculardocking tau ua los txheeb xyuas cov haujlwm ua haujlwm ntawm BID3 hauv cov qauv siv lead ua ntawm nceb tyrosinase (PDB ID: 2Y9X) (Fig. 3A). Cov txiaj ntsig no tau suav nrog siv AutoDock4.2. qhov kev pab cuam. Txoj hauj lwm ruaj khov tshaj plaws yog tias nrog tus qhab nia qis tshaj [50]. Tsis ntev los no, Hassani et al. [47] qhia tias cinnamic acid thiab phthalic acid yog sib xyaw hom tyrosinaseinhibitors. Yog li, kev pab, cinnamic acid, thiab phthalic acid tau siv los ua kom tau txais cov txiaj ntsig docking [51].Cov qauv docking molecular ntawm BID3 thiab kojic acid (zoo-paub kev sib tw hom inhibitor) hauv catalytic site ntawm tyrosinase yog qhia hauv daim duab 3B [52. ]. Lub tyrosinase-BID3 inhibitorcomplex nthuav tawm 6.28 kcal / mol binding zog nrog rau ob daim ntawv cog lus hydrogen nrog ASN260 thiab MET280 residue ntawm tyrosinase, thiab kev sib cuam tshuam hydrophobic tau pom ntawm BID3 thiab tyrosinase residues VAL248, PHE2683, thiab VAL2 kev sib cuam tshuam ntxiv rau qhov chaw. mushroomtyrosinase (Table 2, thiab daim duab 3E thiab H). Ntxiv mus, molecular docking qauv ntawm BID3, phthalic acid (allosteric inhibitor 1), thiab cinnamic acid (allosteric inhibitor 2) nyob rau hauv ob qhov chaw allosteric yog qhia hauv daim duab 3C thiab 3D. Phthalic acid thiab cinnamic acid tau raug coj los ua cov ligands ntawm allosteric sites 1 thiab 2, raws li [47,51]. Raws li pom nyob rau hauv daim duab 3F thiab kuv, BID3 thiab phthalic acid exhibitedone hydrogen daim ntawv cog lus nrog lub TRY140 residue ntawm tyrosinase, thiab peb hydrophobic interactions residues nrog LEU24, PHE147, thiab ILE148 ntawm tyrosinase ntawm allosteric site 1. Cov kev sib cuam tshuam ntawm BID correspons nyob rau hauv allosteric site2 ntawm tyrosinase suav nrog hydrogen bonds ntawm ASP312 thiab LYS376 ntawm tyrosinase, whereas THR308 interacted hydrophobicallyat allosteric site 2 (Fig. 3G thiab J). Tsis tas li ntawd, BID3-tyrosinasebinding tau pom los ua kom muaj zog khi rau hauv ob qho chaw allosteric ntawm tyrosinase (4.38 thiab 5.68 kcal / mol, raws li), qhia tias muaj kev sib raug zoo nrog ob qho chaw allosteric. Raws li kev tshawb fawb enzymekinetic, BID3 tau ua kom muaj kev sib xyaw ua ke, qhov kev sib txuas ntawm BID3 nrog rau ob qho tib si catalytic site thiab ob qhov chaw allosteric tau lees paub nws qhov sib xyaw ua ke ntawm tyrosinase.
Fig. 2. Lineweaver-Burk (A) thiab Dixon plots (B) rau inhibition ntawm nceb tyrosinase los ntawm BID3 siv L-DOPA ua lub substrate.
3.4. Kev ua si lom neeg
Txhawm rau siv cov tshuaj tiv thaiv tyrosinase ntawm BID3 hauv cov qauv kab lis kev cai ntawm tes, peb tau tshuaj xyuas nws cov teebmeem cytotoxic ntawm B16F10 hlwb. Cellswere kho nrog qhov sib txawv ntawm BID3 (1-20 mM) rau 48 h thiab soj ntsuam siv EZ-Cytox assay. Cov txiaj ntsig tau qhia tias BID3 tsis muaj qhov cuam tshuam cytotoxic hauv B16F10 melanomacells txog 20 mM (Fig. 4A thiab B). Yog li, cov kev sim tom qab tau ua tiav siv BID3 txog 20 mM.
Table 2 Kev khi qhov chaw thiab docking cov qhab nia ntawm BID3 hauv nceb tyrosinase (PDB ID: 2Y9X) raws li kev txiav txim siab siv AutoDock4.2 program.
Melanogenesis yog tswj los ntawm tyrosinase enzymatic cascade.Vim li no, ob peb daim tawv nqaij-whitening tebchaw tau tsim los txo cov kev ua ntawm tyrosinase. Kojic acid, tyrosinaseinhibitor, tau siv los ua kev tswj hwm zoo [53]. Yog li, txhawm rau tshawb xyuas qhov cuam tshuam ntawm BID3 ntawm cAMP nce siab induced hyperpigmentation, B16F10 hlwb raug kho nrog a-MSH thiab IBMX nyob rau hauv muaj BID3 (1, 5, thiab 2{{20}} mM) rau 48 h, thiab cov ntsiab lus melanin, thiab cov haujlwm ntawm cellulartyrosinase tau txiav txim siab (Daim duab 4C thiab D). Kev kho mob nrog a-MSH thiab IBMX ua rau muaj kev nce ntxiv (P <0.001#) hauv="" cov="" ntsiab="" lus="" melanin="" (fig.="" 4c)="" thiab,="" cellular="" tyrosinase="" kev="" ua="" haujlwm="" (fig.="" 4d)="" hauv="" b16f10="" hlwb="" piv="" nrog="" kev="" tswj="" tsis="" tau="" kho.txawm="" li="" cas="" los="" xij,="" bid3="" kev="" kho="" mob="" tseem="" ceeb="" (p=""><0.01**; p="">< 0.001***)="" thiab="" concentration-dependently="" txo="" melanin="" cov="" ntsiab="" lus="" thiab="" cellular="" tyrosinase="" kev="" ua="" ntawm="" b16f10="" hlwb="" piv="" nrog="" a-msh="" oribmx="" kho="" hlwb,="" qhia="" tias="" qhov="" txo="" qis="" hauv="" cellular="" melaninmight="" yog="" vim="" qhov="" inhibition="" ntawm="" tyrosinase="" kev="" ua.="" .="" yog="" li,="" cov="" kev="" tshawb="" pom="" no="" qhia="" meej="" meej="" tias="" bid3="" tau="" tawm="" dag="" zog="" los="" tiv="" thaiv="" melanogenic="" cuam="" tshuam="" inhibiting="" tyrosinase="" kev="" ua="" haujlwm="" thiab="" melanin="" synthesis="" hauv="" b16f10="" hlwb="" yam="" tsis="" muaj="" kev="" cuam="" tshuam="">
Indanone yog ib qho ntawm cov qauv tsim nyog ntawm cov tshuaj chemistry thiab feem ntau cuam tshuam nrog ntau yam tshuaj pharmacologicallyactive tebchaw [54]. Lub indanone moiety yog tam sim no nyob rau hauv ntau yam physiologically active khoom ntuj. Raws li qhov no, Okpekonet al., [55] tau tshaj tawm ib qho tshiab 1-indanone compound, yog cais los ntawm Uvaria cov hauv paus hniav. Cov tshuaj no yog thawj 1-indanone derivative cais tawm ntawm cov nroj tsuag. Nagle et al., [56] tau tshaj tawm ib qho tshiab methylated indane-aldehyde raug cais tawm los ntawm marinecyanobacterium raws li cov qog angiogenesis regulator. Ib yam li ntawd, txij li qhov tseem ceeb ntawm lub cev tseem ceeb ntawm indanone core, ntau xyoo dhau los cov kws tshawb fawb tau tsom mus rau kev tsim cov tshuaj pharmacologicallyactive indanone analogs ua cov tshuaj kho mob. Lub structuraldiversity ntawm 1-indanones txhais tau hais tias muaj ntau yam tshuaj lom neeg cov lus teb thiab cov tshuaj no tuaj yeem siv rau hauv kev ua liaj ua teb thiab tshuaj. Ntau yam indanone-based compounds tau tsim los ua cov tshuaj tiv thaiv Alzheimer's disease [16,57–59], anticancer [60– 62], antimicrobial[63,64], thiab tshuaj tiv thaiv kab mob [65,66]. Vim muaj lub peev xwm siv dav dav, 1-indanones yog cov khoom nthuav dav rau kev tshawb fawb ntxiv thiab nws yog qhov tsim nyog los tsim cov chav kawm tshiab rau lawv cov kev sib txuas.
cistanche tubolosa: tiv thaiv kev laus
4. Cov lus xaus
Txhawm rau txheeb xyuas cov muaj zog tyrosinase inhibitors tshiab, peb tau tsim thiab ua ke kaum peb (E)-benzylidene-1-indanone derivatives.Ntawm cov tshuaj no, (E)-2-(2,4-dihydroxy-benzylidene){ {6}}, 3-dihydro-1H-in{10}}ib (BID3) muaj zog inhibited tyrosinase kev ua haujlwm (IC50=0.034 thiab 1.39 mM, rau L-tyrosine thiab L-DOPA, ntsig txog), uas zoo dua li cov ntaub ntawv siv, kojic acid (IC50=13.77 mM thiab 33.14 mM, feem). Cov qauv kev sib raug zoo-kev sib raug zoo tau qhia tias muaj cov pab pawg dihydroxyl ntawm 2 thiab 4- txoj hauj lwm ntawm (E)-benzylidene-1-indanone skeletonimproved cov kev ua ntawm tyrosinase. Hom ntawm enzymaticinhibition ntawm cov enzyme, txiav txim los ntawm Lineweaver-Burk thiab Dixon cov phiaj xwm, qhia tias BID3 yog ib hom tshuaj sib xyaw. Kev tshawb fawb molecular docking qhia tau hais tias kev khi ntawm qhov chaw nquag thiab ntawm cov chaw allosteric yog cov txheej txheem tseem ceeb rau kev ua haujlwm ntawm tyrosinase inhibitory. Raws li cov txiaj ntsig no, BID3 zoo li yog lub zog tyrosinase inhibitor rau kev kho mob ntawm daim tawv nqaij xws li hyperpigmentation.
Cov txiaj ntsig ntawm cov suab puam cistanche: inhibits melanin tsim
