ABSTRACT

Keeb kwm:Ib qho bis resorcinol raug cais raws li lub ntsiab lus tseem ceeb ntawm Heliciopsis terminals 'strunk (Proteaceae). Tsis ntev los no, resorcinol tau siv los ua tus neeg sawv cev dawb huv hauv ntau yam khoom siv tshuaj pleev ib ce. Vim hais tias ntawm cov qauv qauv ntawm resorcinol, cov txiaj ntsig ntawm bis resorcinol raws li kev laus-enzyme antagonist tau pom nyob rau hauv txoj kev tshawb no.

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.

Nyem rau ntawm Cistanche Tubulosa rau Whitening

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Txoj kev:Lub bis resorcinol tau purified los ntawm crude ethanolic extract ntawm H. terminals lub cev los ntawm hnyav extraction thiab npaj chromatography, feem. Inhibitory kev ua ntawm collagenase, elastase, thiab tyrosinase ntawm lub compound tau tshawb xyuas los ntawm kev siv cov txheej txheem spectroscopic sib txawv. Molecular docking tau ua tiav los kwv yees kev sib cuam tshuam ntawm cov khoom nyob ib puag ncig cov enzyme active chaw.

Cov txiaj ntsig:IC50 qhov tseem ceeb ntawm collagenase ntawm bis resorcinol thiab caffeic acid yog 156.7 ± 0.7 thiab 308.9 ± 1.6 µmole L-1, raws li. Rau kev ua elastase, IC50 ntawm 33.2 ± 0.5 thiab 34.3 ± 0.3 µmole L-1 tau raug txiav txim siab raws li bis resorcinol thiab ursolic acid. Lub bis resorcinol yog inhibitory rau tyrosinase los ntawm kev nthuav tawm IC50 ntawm 22.8 µmole L-1, thiab ntawm 78.4 µmole L-1 yog tam sim no rau -arbutin. Lub bis resorcinol khi rau collagenase, elastase, thiab tyrosinase nrog rau kev sib txuas zog ntawm −5.89, −5.69, thiab −6.57 kcal mol-1. Cov binding zog no nyob rau tib yam li kev sim inhibitors. Cov pab pawg muaj ntxhiab phenol nyob rau hauv cov qauv yog lub luag haujlwm rau lub hauv paus ntsiab lus nrog rau kev txhawb nqa kev sib txuas nrog cov enzymes. Hydrogen binding vim yog pawg hydroxyl thiab π-txog kev txaus nyiam zog los ntawm ib lub nplhaib uas muaj ntxhiab muab kev sib txuas ntau yam rau bis resorcinol.

Xaus:Lub bis resorcinol purified los ntawm H. terminals tej zaum yuav muaj txiaj ntsig rau kev suav nrog hauv cov khoom siv tshuaj pleev ib ce raws li kev laus-enzyme antagonist.

Cov kawm Biochemistry, Bioinformatics, Cell Biology, Dermatology, Data Mining, thiab Machine Learning

Ntsiab lusHeliciopsis terminals, Bisresorcinol, Collagenase, Elastase, Tyrosinase, Anti-aging khoom, Molecular docking, Enzyme inhibition

Taw qhia

A bis resorcinol, (8′Z)-3,5-dihydroxy-1-[16′-(3″, 5″-dihydroxy phenyl)-8}′- hexadecen{{ 11}}′-yl] benzene, tau raug cais tawm raws li cov khoom siv ntau tshaj plaws los ntawm lub cev ntawm Heliciopsis terminals (Kurz) Sleumer, uas yog cov nroj tsuag ntawm tsev neeg Proteaceae thiab tau siv los ntawm cov neeg nyob hauv Nyab Laj rau cov nyhuv hepatoprotective. Txog niaj hnub no, cov txiaj ntsig kho mob ntawm cov tshuaj no ua tus neeg sawv cev rau kev tiv thaiv kab mob, inhibition ntawm malondialdehyde (MDA) ntau lawm, thiab hepatoprotection tau qhia meej hauv vitro thiab hauv vivo (Giang li al., 2019). Nws pom tseeb tias ib qho molecule ntawm bis resorcinol muaj ob lub resorcinol molecules (EC No 203-585-2), sib koom ua ke nrog meta-C7H14C=CC7H14 linkage (Fig. 1A). Resorcinol tsis ntev los no tau siv rau hauv daim ntawv ntawm lub ntsej muag concentrated serum rau kev kho dawb. Raws li Pawg Neeg Saib Xyuas Kev Nyab Xeeb Kev Noj Qab Haus Huv, Brussels, Belgium (SCCS 1270/09, European Commission, Scientific Committee on Consumer Safety, 2010), resorcinol tau raug tswj los ua cov oxidant hauv cov khoom npaj rau xim plaub muag ntawm cov concentration ntau txog 1.25 feem pua. , los yog nyob rau hauv cov plaub hau tshuaj pleev ib ce thiab tshuaj zawv plaub hau nyob rau hauv concentration ntau txog 0.5 feem pua. Kev siv tshuaj pleev ib ce ntawm resorcinol muaj ntau yam. Rau cov neeg ua kom dawb, inhibition ntawm melanin synthesis los ntawm melanocytes uas nyob rau hauv lub basal cell txheej ntawm lub epidermis tau raug suav tias yog. Tyrosinase (EC 1.14.18.1) yog ib qho tseem ceeb enzyme rau catalyzing melanogenesis uas L-tyrosine yog siv los ua substrate. Txawm hais tias melanin pab tiv thaiv daim tawv nqaij los ntawm kev puas tsuaj los ntawm UV rays, ntau melanin ntau lawm yuav ua rau hyperpigmentation, freckle, thiab hnub nyoog me ntsis. Ntxiv nrog rau resorcinol, ntau yam hluavtaws thiab ntuj sib xyaw nrog cov qauv tshuaj sib txawv tau pom tias muaj kev ua haujlwm los tiv thaiv tyrosinase (Dobos li al., 2015).

UV hluav taws xob yog qhov tseem ceeb sab nraud uas ua rau tawv nqaij laus. Hloov chaw, cov tawv nqaij tuaj yeem muaj hnub nyoog dhau sijhawm los ntawm cov pa oxygen reactive (ROS) thiab lipid peroxides uas tsim tawm sab hauv tom qab UV raug. Cov khoom thib ob los ntawm lipid peroxide metabolism tuaj yeem ua rau elastin thiab collagen fibers ntawm extracellular matrices (ECMs). Yog li ntawd, raug UV rays tuaj yeem ua rau lub cev qhuav dej, poob elasticity, nrog rau wrinkles ntawm daim tawv nqaij. Rau peb txoj kev paub, ECMs tau hloov kho ib ntus kom tswj cov ntaub so ntswg ib txwm ua nrog rau cov ntaub so ntswg uas tas sij hawm tau raug degraded los ntawm matrix metalloproteinases, xws li collagenases, thiab serine proteases zoo li elastases (Thring, Hili & Naughton, 2009). Tsis tas li ntawd, ob qho tib si ntau thiab cov dej num ntawm cov enzymes laus tau nce los ntawm oxidative kev nyuaj siab, ua rau ECM degradation thiab kev laus zuj zus (Sherratt li al., 2019). Yog li ntawd, cov tebchaw uas tuaj yeem tsim cov complexes nrog cov hlau ions tau cia siab tias yuav inhibitory rau collagenases, elastases, thiab tyrosinase raws li (Selvaraj li al., 2014). Qhov phiaj xwm no yog txhawm rau txiav txim siab txog cov txiaj ntsig lom neeg ntawm bis resorcinol ntawm tyrosinase, collagenase, thiab elastase hauv vitro, thiab cov txheej txheem docking tau ua kom paub meej lawv cov kev sib cuam tshuam ntawm qib molecular. Nws cia siab tias yuav ncua kev laus thiab txhim kho daim tawv nqaij nrog bis resorcinol.

MATERIALS & METHODS

Tshuaj thiab cov cuab yeej siv

Compound purification, txheeb xyuas, thiab pom zoo

Cov kua txiv hmab txiv ntoo hauv ethanol ntawm H. terminals lub cev tau txais txiaj ntsig zoo los ntawm xibfwb PM Giang, Kws Qhia Ntawv ntawm Chemistry, Vietnam National University, Hanoi, Nyab Laj nrog daim ntawv pov thawj tus lej HNIP-18473. Tom qab ntawd, nws tau muab faib rau hauv methanol / n-hexane los ntawm peb zaug dua. Cov txheej methanol tau evaporated kom qhuav thiab tom qab ntawd muab faib rau hauv ethyl acetate/1-butane. ethyl acetate txheej tau vaporized kom txog thaum nws ntim tau txo los ntawm 80 feem pua. Cov kua nplaum uas tau tshwm sim tau raug ntxuav ntawm silica gel kem siv chloroform / methanol gradient elution raws li hauv qab no: 100 feem pua ​​​​chloroform, 30: 1, 20: 1, 10: 1, 7: 1, 5: 1, 3: 1, 3 : 2, thiab 2: 1 ntawm chloroform / methanol, thiab 100 feem pua ​​​​methanol, feem. Cov eluent los ntawm 5: 1 tov cov kuab tshuaj tau sau thiab evaporated rau ib-plaub ntim, tom qab ntawd los ntawm kev sib cais ntawm ODS kem siv gradient elution ntawm methanol thiab acetone. Cov khoom sib xyaw uas xav tau, piv txwv li, bis resorcinol (Fig. 1), tau txais los ntawm cov khoom muaj 70 feem pua ​​​​acetone thiab lees paub los ntawm MS, NMR, thiab IR spectroscopies txog cov ntaub ntawv hauv tsev qiv ntawv. Nyias-txheej chromatography tau siv rau kev kwv yees ntawm nws cov purity.

Kev ntsuam xyuas kev ua haujlwm lom neeg

Hauv vitro, kev sim uas muaj enzyme-inhibitory kev soj ntsuam ntawm collagenase, elastase, thiab tyrosinase rau bis resorcinol thiab paub inhibitors tau ua raws li hauv qab no.

Collagenase inhibition

Qhov kev ntsuam xyuas anti-collagenase tsis ntev los no tau hloov kho los ntawm qhov tau piav qhia yav dhau los los ntawm Widyowati li al. (2016). Hauv kev luv luv, 50 mmol L-1 tricine tsis pH 7.5 ntxiv nrog 400 mmol L-1 NaCl thiab 10 mmol L-1 CaCl2 tau siv los ua cov buffering diluent. Collagenase los ntawm Clostridium histolyticum (EC.3.4.24.3) tau yaj hauv qhov tsis sib xws rau qhov concentration ntawm 1 mg L-1. Cov enzyme-substrate, MOCAc-PRO-Leu-Gly-Leu-A2pr (Dnp)-Ala-Arg-NH2, tau ua ntej yaj hauv DMSO thiab tom qab ntawd diluted hauv qhov tsis txaus mus rau qhov siab ntawm 1 mmol L-1. Ib qho piv txwv yaj hauv DMSO tau diluted rau ntau qhov sib txawv los ntawm kev siv qhov tsis. Ib qho 2-µl cov qauv tshuaj tau tsim nrog 100 µl enzyme tov ntawm 37 ○C rau 10 min. Tom qab ntawd, ib qho 50-µl substrate tau ntxiv thiab sib xyaw kom zoo. Fluorescence emission (F) ntawm 405 nm tau kaw tam sim ntawd thiab txuas ntxiv saib xyuas rau 30 min siv lub wavelength ntawm 320 nm rau kev zoo siab. Caffeic acid tau siv los tswj txoj haujlwm (Fig. 1). Kev tswj tsis zoo tau ua nrog dej. Qhov feem pua ​​inhibition (%) yog xam los ntawm qhov sib npaug hauv qab no:

feem pua ​​Inhibition {{0}}{1 - (Fsam; 30 - Fsam; 0)÷(Fcont; 30 - Fcont; 0)} × 100.

Kev tiv thaiv elastase

Qhov kev ntsuam xyuas raws li Abhijit & Manjushree (2010) tau siv nrog qee qhov kev hloov kho. Luv luv, lub kaw lus tsis yog 0.2 mM Tris-HCl tsis (pH 8.0). Kev daws ntawm 1 µg mL-1 porcine pancreatic elastase (EC.3.4.21.36) tau npaj rau hauv dej tsis muaj menyuam. Cov enzyme substrate, N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), tau yaj hauv qhov tsis pom kev mus rau qhov siab ntawm 80 mmol L-1. Ib qho piv txwv yaj hauv DMSO tau diluted mus rau ntau qhov ntau los ntawm kev siv qhov tsis. Ib qho piv txwv {{20}µl tov nrog 50 µl enzyme tov thiab incubated rau 15 feeb. Tom qab ntawd 50 µl ntawm cov tshuaj enzyme-substrate tau ntxiv thiab sib xyaw kom zoo. OD410 tau ntsuas tam sim ntawd (0 min) thiab tom qab incubation thaum hmo ntuj (o / n) ntawm 37 degree los ntawm kev siv lub microplate nyeem ntawv. Ursolic acid tau siv los ua kev tswj xyuas qhov zoo (Daim duab 1), thiab dej tau siv los ua kev tswj tsis zoo. Qhov feem pua ​​inhibition (feem pua) tau xam los ntawm qhov sib npaug hauv qab no:

feem pua ​​Inhibition {{0}} {1 - [(Asam; o/n - Asam; 0)÷(Acont; o/n - Acont; 0)]} × 100:

Tyrosinase inhibition

Kev siv tyrosinase inhibitory assay tau hloov kho los ntawm txoj kev piav qhia yav dhau los (Jiratchayamaethasakul li al., 2020). Hauv luv luv, qhov kev ntsuam xyuas tau ua nyob rau hauv 0.05 mol L−1 phosphate tsis pH 6.8. Mushroom tyrosinase (EC.1.14.18.1) tau yaj hauv qhov tsis sib haum mus rau qhov siab ntawm 100 units mL-1. L-tyrosine, ib qho enzyme substrate, tau npaj rau qhov concentration ntawm 0.25 mg mL-1 hauv qhov tsis muaj. Ib qho kev sim tshuaj yaj hauv DMSO tau diluted mus rau qhov siab ntawm 0.625 thiab 10 mg mL-1. Nyob rau hauv ib lub qhov dej ntawm 96-zoo daim hlau, ib tug 10 µl qauv thiab 40 µl L-tyrosine tov thiab incubated rau 10 min nyob rau hauv chav tsev kub. Tom qab ntawd 50 µl enzyme tov yog inoculated thiab sib tov kom huv si. Cov tshuaj tiv thaiv tau nres tom qab los ntawm incubation ntawm dej khov rau 1 min. OD475 tau ntsuas los ntawm kev nyeem ntawv microplate. -Arbutin thiab dej tau siv los ua kev tswj xyuas qhov zoo (Daim duab 1) thiab cov khoom tsis zoo, feem. Qhov feem pua ​​inhibition (%) yog xam los ntawm qhov sib npaug raws li hauv qab no:

MOLECULAR DOCKING STUDY

Kev npaj ntawm ligands thiab receptors

Peb-dimensional (3D) cov qauv ntawm bis resorcinol (CID 8917124) thiab cov ntaub ntawv siv, suav nrog cov substrates thiab inhibitors, tau rub tawm los ntawm PubChem database rau kev tshawb fawb molecular docking. Cov qauv ligand tau npaj rau hauv Protein Data Bank (PDB) cov ntaub ntawv siv online SMILES Translator thiab Structure File Generator. Cov qauv siv lead ua ntawm cov enzymes, xws li collagenase (PDB ID 2Y6I), elastase (PDB ID 1BRU), thiab tyrosinase (PDB ID 2Y9X) tau txais los ntawm RCSB protein database. Cov dej thiab cov molecules txuas tau raug txiav tawm los ntawm tag nrho cov txheej txheem protein. Lub caij no, cov polar hydrogen atoms tau ntxiv rau cov qauv protein crystallized los ntawm AutoDockTools 1.5.6. Cov ntaub ntawv ntawm lub hom phiaj cov protein thiab lwm cov khoom siv tau raug cawm hauv PDBQT hom ua ntej ua cov khoom siv molecular.

Xaiv qhov chaw seem seem thiab molecular docking

Daim phiaj thiab docking raws tu qauv ntawm qhov chaw ua haujlwm kwv yees tau npaj siv AutoDockTools 1.5.6. Daim phiaj chaw tau teeb tsa nrog qhov sib nrug ntawm 0.375 Å. Lub xyz qhov ntev tau teem rau 126-135-160 Å3 rau collagenase, 130-120-126 Å3 rau elastase, thiab 80-80-80 Å3 rau tyrosinase. Grid box centers (nrog rau cov nqi offset hauv AutoDockTools) yog 31.869 (5.000), −19.41 (−25.{20}}), thiab 17.815 rau collagenase , 30.048 (−0.972), 51.253 (−2.722), 17.6 rau elastase, thiab −8.407 (−1.{36}}), −23.795 (−0.250), −36.019 (−3.500) ) rau tyrosinase, feem. Cov qauv protein tau siv los ua ib qho chaw nruj thaum lub ligand compound tau teeb tsa raws li cov qauv hloov pauv. Txoj kev tshawb fawb docking tau ua tiav siv Lamarckian genetic algorithm (GA) siv los ntawm AutoDock4 version 4.2. Tus naj npawb ntawm GA khiav yog 50 nrog qhov nrov loj ntawm 200. Lub zog khi (ΔGbind) tau soj ntsuam los ntawm ADT. Kev sib tham (s) tau pom pom siv BIOVIA Discovery Studio (BIOVIA, 2020). Kev sib cuam tshuam ntawm molecular ntawm cov khoom sib xyaw thiab cov protein receptor tau txheeb xyuas thiab pom pom siv BIOVIA Discovery Studio software (BIOVIA, 2020). Cov qauv ntawm cov protein khi qhov chaw sib txuas tau pom pom siv pob Visual Molecular Dynamics (VMD) (Humphrey, Dalke & Schulten, 1996).

Kev txheeb cais

TSEEM CEEB

Ib qho tseem ceeb tau ua tiav huv huv los ntawm H. terminals lub cev los ntawm kev siv cov kuab tshuaj rho tawm thiab thim rov qab cov txheej txheem HPLC, feem. Qhov tau txais 1 H-NMR thiab 13C-NMR spectra tau muab piv rau cov ntaub ntawv siv los ntawm SciFinder (https://scifinder.cas.org/) thiab pom tias sib haum nrog (8′Z)-1,{ {6}}dihydroxy-5- [16′-(3″,5″-dihyroxyphenyl)-8′-hexadezen-1}′-yl] benzene (Chaturvedula li al., 2{{ 23}}02). Nws tau muab cais ua ib qho ntawm cov bisresorcinols. Qhov no bis resorcinol tsis ntev los no muaj cov qauv molecular ntawm C28H40O4 nrog ib qho hnyav molecular ntawm 463.2819 Dalton. Kev rho tawm tawm tau suav tias yog 0.086 feem pua, raws li lub pob tw 'qhuav hnyav. Nws purity yog ntau tshaj 98 feem pua, hais txog 1 H thiab 13C-NMR spectra tau txais (saib Fig. S1 thiab S2).

Hauv vitro bioactivity tsom xam

Cov teebmeem antagonistic ntawm bisresorcinol ntawm cov enzymes laus, xws li collagenase, elastase, thiab tyrosinase tau txiav txim siab hauv vitro los ntawm kev siv txoj kev sib txawv spectroscopic. Cov txiaj ntsig tau muab sau tseg hauv Table 1 thiab Fig. 2. Rau collagenase inhibition, kuaj cov qauv hauv thaj tsam ntawm 50–550 µmol L−1 tau npaj siv MOCAc-PRO-Leu-Gly-Leu-A2pr (Dnp)-Ala Arg-NH2 raws li enzyme-substrate thiab caffeic acid ua ib qho enzyme inhibitor. Nws tau qhia tias bisresorcinol ntawm qhov concentration ntawm 156.7 µmol L-1 txo cov enzyme kev ua haujlwm los ntawm 50 feem pua ​​(IC50). Hloov chaw, IC50 ntawm caffeic acid yog 308.9 ± 13.7 µmol L-1. Yog li, kev ua haujlwm los tiv thaiv collagenase ntawm bisresorcinol muaj zog ntau dua li caffeic acid.

Yog xav paub ntxiv: david.deng@wecistanche.com WhatApp:86 13632399501