Kev laus ntawm daim tawv nqaij yog ib qho teeb meem lom neeg tshwm sim vim qhov physiological txo qis

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Abstract:Lub hom phiaj ntawm txoj kev tshawb no yog txhawm rau kuaj cov nyhuv inhibitory ntawm cov txiv hmab txiv ntoo extracts los ntawm Washingtonia filifera ntawm daim tawv nqaij aging-related enzymes. Cov kua txiv hmab txiv ntoo tsis tau ua kom muaj zog enzyme inhibition thaum cov noob rho tawm los ntawm W.filifera nthuav tawm anti-elastase, anti-collagenase, thiab anti-tyrosinase kev ua ub no. Tyrosinase tau me ntsis inhibited thaum lub zog muaj zog tau pom nrog kev hwm rau elastase thiab collagenase inhibition. Alcoholic extracts muab cov txiaj ntsig zoo dua li aqueous extracts. Ntawm lawv, cov tshuaj methanol tau pom cov enzyme tseem ceeb inhibitory kev ua ub no yog IC5o tus nqi rau elastase thiab collagenase piv thiab txawm zoo dua li cov ntaub ntawv siv. Txoj kev inhibition ntawm cov tshuaj nquag tshaj plaws tau tshawb xyuas los ntawm Lineweaver-Burk kev tsom xam. Cov noob rho tawm los ntawm W.filifera kuj tau tshawb xyuas rau lawv cov kev tiv thaiv kev yees duab los ntawm Mansur equation thiab kev ua haujlwm antioxidant ntawm W.filifera extract tau soj ntsuam hauv oxidative-stressed hlwb. Txhawm rau ntsuas kev nyab xeeb ntawm cov extract, cov nyhuv ntawm cov cell viability ntawm tib neeg keratinocytes hlwb raug tshuaj xyuas.poob teb chaws Ottoman cistancheMethanol extract tau nthuav tawm qhov zoo tshaj plaws yees duab tiv thaiv cov nyhuv thiab ua haujlwm antioxidant hauv lub cellular system uas tsis muaj cytotoxic nyhuv. Cov txiaj ntsig tag nrho qhia tau hais tias W.filifera extracts yog cov chaw cog lus ntawm bioactive compounds uas tuaj yeem siv rau hauv tshuaj pleev ib ce thiab tshuaj npaj.

Ntsiab lus:inhibition ntawm enzymes; collagenase; elastase; tyrosinase; cog extracts; noob; tawv nqaij laus; Washingtonia filifera

Thov nias ntawm no kom paub ntxiv

1. Taw qhia

Kev laus ntawm daim tawv nqaij yog ib qho tshwm sim ntawm cov kab mob lom neeg vim yog lub cev tsis muaj zog ntawm daim tawv nqaij ua haujlwm thiab ntau qhov chaw nyob ib puag ncig, xws li UV hluav taws xob, tshuaj lom neeg, thiab hom pa oxygen reactive (ROS). Daim tawv nqaij yog qhov loj tshaj plaws thiab feem ntau nthuav tawm ntawm lub cev thiab raug rau hnub ci UV hluav taws xob sawv cev yog ib qho tseem ceeb tshaj plaws sab nraud kev ntxhov siab-inducing yam: yees duab-induced tawv nqaij laus txuam nrog oxidative kev nyuaj siab. ROS induced los ntawm UV irradiation tuaj yeem pib txoj hauv kev yooj yim molecular, suav nrog kev ua kom cov enzymes uas degrade extracellular matrix (ECM) cov proteins hauv dermis, hloov cov tawv nqaij kev ncaj ncees [1]. Ib qho ntawm cov yam ntxwv tseem ceeb ntawm daim tawv nqaij laus yog qhov tseeb Los ntawm cov qauv ntawm ECM, uas muaj ntau cov proteins, suav nrog collagen thiab elastin, tag nrho cov uas ua lub luag haujlwm tseem ceeb hauv kev khaws cov tawv nqaij elasticity [2]. Kev puas tsuaj ntawm ECM feem ntau yog vim muaj kev txhim kho ntawm cov enzymes proteolytic, xws li collagenase thiab elastase. Inhibition ntawm cov enzymatic kev ua ub no los ntawm tej yam ntuj tso nroj tsuag tebchaw tej zaum yuav ib tug cog lus mus kom ze rau tiv thaiv daim tawv nqaij laus [3]. Collagenase (EC 3.4.24.3) belongs rau tsev neeg ntawm matrix metalloproteinases thiab nws muaj peev xwm degrades lub triple-helical cheeb tsam ntawm collagen nyob rau hauv physiological mob.micronized purified flavonoid feem 1000 mg sivCollagen yog cov khoom siv fibrous ntawm ECM thiab cov txheej txheem tseem ceeb hauv tib neeg cov tawv nqaij, muab kev txhawb nqa rau cov pob txha, leeg, ligaments, thiab cov hlab ntsha. Elastase (EC3.4.21.36) yog ib qho enzyme proteolytic koom nrog hauv lub cev degradation ntawm elastin, ECM protein ua lub luag haujlwm rau daim tawv nqaij elasticity. Kev nce ntawm elastase kev ua haujlwm tau pom nyob rau hauv ntau yam kab mob, piv txwv li, psoriasis, dermatitis, inflammatory txheej txheem, thiab cov tawv nqaij laus ntxov ntxov, uas muaj feem cuam tshuam nrog kev tsim ntawm wrinkles [4].

Cistanche tuaj yeem tiv thaiv kev laus

Ntxiv mus, ib qho ntawm cov kev hloov loj cuam tshuam nrog wrinkling nyob rau hauv cov neeg laus yog cov tsos ntawm hyperpigmented me ntsis, kuj hu ua senile lentigo los yog hnub nyoog me ntsis. Lawv ncaj qha cuam tshuam nrog cov pigmentation tsis sib xws vim kev ua haujlwm ntawm lwm cov enzyme uas muaj npe hu ua tyrosinase. Tyrosinase (EC 1.14.18.1) yog tus nqi txwv enzyme hauv cov metabolism ntawm melanin. Nws catalyzes hydroxylation ntawm L-tyrosine rau 3, 4-dihydroxyphenylalanine (L-DOPA), tom qab ntawd oxidation ntawm L-DOPA rau dopaquinone. Oxidative polymerization ntawm dopaquinone derivatives ua rau muaj melanin [5]. Lub synthesis ntawm melanin pigments yog txheej txheem physiological uas ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv UV-induced tawv nqaij puas los ntawm absorbing UV tshav ntuj. Txawm hais tias nws qhov zoo, kev tsim khoom ntau dhau los yog qhov txawv txav ntawm melanin ua rau muaj teeb meem ntawm daim tawv nqaij xws li cov hnub nyoog ib txwm muaj. Txij li thaum daim tawv nqaij yog qhov pom kev tshaj plaws ntawm lub cev, qhov tshwm sim ntxov ntxov ntawm wrinkles thiab hyperpigmentation tuaj yeem ua rau muaj kev ntxhov siab rau qee tus neeg.

Yog li, inhibitors ntawm tag nrho cov saum toj no-hais enzymes yuav sawv cev rau cov khoom xyaw tseem ceeb nyob rau hauv cov tshuaj pleev ib ce thiab tshuaj tiv thaiv daim tawv nqaij laus [6]. Cov khoom cog ntoo tuaj yeem yog qhov cog lus ntawm bioactive compounds. Ntawm qhov kev txaus siab tshwj xeeb rau kev siv tshuaj tiv thaiv kev laus, cov extracts muaj ntau yam txiaj ntsig zoo, xws li inhibition ntawm aging-txog enzymes thiab lub peev xwm ntawm scavenging dawb radicals.

Hauv peb txoj haujlwm dhau los, peb tau piav qhia txog lub peev xwm antioxidant thiab ntau yam kev ua haujlwm lom neeg ntawm W.filifera noob rho tawm [7]. Washington filifera (Lindl.) H. Wendl., feem ntau hu ua California kiv cua xibtes lossis suab puam kiv cua xibtes, yog ib tsob ntoo tsob ntoo ntsuab uas nyob rau yav qab teb California, Arizona, Mexico, thiab thaj chaw suab puam.oteflavonoidLub xibtes no, nrog qhov siab ntawm 15-20 m, tsis tsim hnub tab sis muaj cov txiv hmab txiv ntoo qab zib thiab qab qab. Cov txiv hmab txiv ntoo no muaj cov noob loj heev, xim av ncig los ntawm nyias nyias (Daim duab 1). W.filifera tau kawm txog, piv txwv li, nws siv los ua lub peev xwm ntawm cov cellulosic fibers tshiab [8] thiab cov khoom noj muaj txiaj ntsig ntawm nws cov txiv hmab txiv ntoo [9]; cov phenolic muaj pes tsawg leeg thiab kev ua haujlwm antioxidant ntawm lub dav hlau qhov chaw muaj leen tau tshaj tawm [10]. Hauv peb txoj kev tshawb fawb dhau los, peb tau tshaj tawm cov kev ua haujlwm zoo antioxidant ntawm W.puritans vitamin cfilifera noob rho tawm, uas tshwm sim los ntawm phenolic thiab flavonoid molecules[7]. Tib cov extracts exerted ib inhibitory nyhuv ntawm xanthine oxidase thiab cholinesterase enzymes, uas sawv cev rau cov enzymes tseem ceeb nyob rau hauv kev kho mob ntawm gout thiab Alzheimer's kab mob, feem.

Lub hom phiaj ntawm txoj haujlwm no yog txhawm rau txuas ntxiv tus yam ntxwv ntawm cov nroj tsuag no thiab tshuaj xyuas qhov muaj peev xwm siv nws cov extracts ua tus neeg sawv cev tiv thaiv kev laus. Pulp thiab noob rho tawm tau, yog li ntawd, tshawb xyuas rau lawv cov haujlwm inhibitory rau elastase, collagenase, thiab tyrosinase kev ua haujlwm, vim tias lawv sawv cev rau lub hom phiaj tseem ceeb rau kev tiv thaiv thiab kho ntawm daim tawv nqaij photoaging; Ntxiv mus, W.filifera extracts uas qhia txog qhov zoo tshaj plaws kev ua ub no tau soj ntsuam rau lawv nyob rau hauv vitro cytotoxicity, cellular antioxidant kev ua, thiab yees duab-tiv thaiv teebmeem.

2. Cov txiaj ntsig thiab kev sib tham

2.1.Enzyme Inhibition

W. filifera pulp thiab noob rho tawm tau kuaj thawj zaug ntawm qhov concentration ntawm 50 ug / mL.sibTag nrho cov pulp extracts tsis exert tseem ceeb enzyme inhibition (cov ntaub ntawv tsis qhia). Cov enzyme inhibitory kev ua ub no ntawm cov noob rho tawm tau suav thiab qhia tias yog ib nrab-qhov siab tshaj plaws inhibitory concentration (tseem). Table 1 qhia txog ICso qhov tseem ceeb tau los ntawm cov noob rho tawm piv nrog cov txheej txheem inhibitors, txhawm rau ntsuas qhov inhibitory zog ntawm cov qauv. Raws li nws tuaj yeem pom, tag nrho cov extracts tsis muaj zog tiv thaiv kev ua haujlwm ntawm tyrosinase, nrog ICso cov txiaj ntsig siab dua li cov qauv, kojic acid. Kev tiv thaiv zoo dua tau pom tawm tsam elastase thiab collagenase kev ua ub no, thiab ethanolic (EEG thiab EES) thiab methanolic (MEG thiab MES) rho tawm cov txiaj ntsig zoo tshaj plaws. Kev ua haujlwm inhibitory ntawm cov qauv no tiv thaiv elastase zoo ib yam; ICso qhov tseem ceeb nyob ntawm 10.75 txog 19.75 ug / mL thiab tau muab piv rau cov kev tswj zoo (oleanolic acid; ICso=11.75 ug/mL).

Hloov chaw, collagenase kev ua tau zoo inhibited los ntawm cov extracts, uas pom tias muaj zog ntau dua li tus qauv epigallocatechin gallate, ICso tau nce mus txog peb npaug qis dua qhov kev tswj zoo. Muab hais tias cov extracts yog ib tug sib tov ntawm ob peb lub tebchaw, lub concentration ntawm ib tug active molecules yog txawm qis dua tus nqi IC50, yog li ua rau cov extracts txawm ntau pheej qhov chaw ntawm inhibitory tebchaw.

2.2.Kinetic Analysis los ntawm Lineweaver-Burk Plot

Peb tsom peb cov xim rau ethanol thiab methanol rho tawm txhawm rau tshawb xyuas hom inhibition ntawm cov enzymes vim tias lawv muaj kev cuam tshuam zoo dua rau cov dej num elastase thiab collagenase. Lub kinetic ntawm inhibition tau txiav txim siab los ntawm Lineweaver-Burk ob chav sib txuas lus. Cov kev ntsuam xyuas tau ua los ntawm kev nce qhov concentration ntawm cov substrate nyob rau hauv tsis muaj thiab muaj cov extracts ntawm txawv concentrations.

Table2 qhia tau hais tias EEG thiab EES ua raws li qhov tsis sib haum xeeb inhibitors tiv thaiv elastase. Nyob rau hauv qhov tseeb, lub kinetic tsom xam ntawm cov extracts tau tsim ib tsev neeg ntawm cov kab sib txuas rau kev nce extract concentrations (Daim duab 2A, B).Qhov kev tsom xam kiretic qhia tias cov extracts tuaj yeem khi nrog cov enzyme-substrate complex. Qhov sib npaug tsis tu ncua (Krs) tau suav los ntawm kev rov ua dua ntawm kev cuam tshuam (1 / Vmax) piv rau inhibitor concentration, ua rau tus nqi ntawm 3.91 thiab 8.89 ug / mL rau EEG thiab EES, feem. Hom ntawm inhibition ntawm methanolic extracts tseeb qhia tau hais tias cov extracts ua raws li ib tug noncompetitive inhibitor. Qhov tseeb, los ntawm kev nce cov concentration ntawm cov extracts, ib tsev neeg ntawm cov kab ncaj nraim nrog cov kab sib txawv, txhua qhov kev sib tshuam ntawm abscissa, tau pom (Daim duab 2C, D). Qhov kev soj ntsuam no qhia tau hais tias cov extracts tuaj yeem khi tsis tau tsuas yog rau cov enzyme-substrate complex tab sis kuj rau cov enzyme dawb. Qhov sib npaug sib npaug rau kev khi nrog cov enzyme dawb (Kj) thiab nrog cov enzyme-substrate complex (Kis) tau txais los ntawm txoj kab nqes (Km / Vmax) lossis 1 / Vmax qhov tseem ceeb (y-intercepts) plotted piv rau inhibitor concentration, raws.Thaum kawg, tus cwj pwm kinetic ntawm collagenase ntawm qhov sib txawv ntawm cov extracts yog pom hauv daim duab 3A-D. Tag nrho cov extracts ua raws li kev tsis sib haum xeeb inhibitors nrog Krs qhov tseem ceeb hauv thaj tsam ntawm 7.58-13.04 ug/mL (Table 2).

Hauv peb txoj haujlwm dhau los, peb tau txheeb xyuas cov kua dej cawv ntawm W. filifera noob, siv HPLC-DAD-ESI/MS. Peb tau hais tias lub ntsiab phenolic tebchaw ntawm cov extracts muaj flavan-3-ol [7]. Ntawm lawv, B-hom procyanidins yog cov ntsiab lus tseem ceeb hauv kev rho tawm ntawm W.filifera noob. Kev sib raug zoo ntawm qib ntawm procyanidin polymerization thiab lub peev xwm ntawm procyanidins los inhibit elastase tau pom nyob rau hauv daim ntawv dhau los [11,12]. Tsis tas li ntawd, inhibitory kev ua haujlwm tawm tsam elastase thiab collagenase los ntawm qee cov tshuaj procyanidin tau tshaj tawm [13,14]. Kev sib koom ua ke ntawm cov tebchaw no tuaj yeem pab piav qhia qhov tseem ceeb inhibition ntawm W.filifera methanolic extract tawm tsam ob lub enzymes.

2.3.Sun Protection Factor

Photoprotectant kev ua si yog ib qho tseem ceeb heev rau cov tebchaw nrog rau daim tawv nqaij daim ntawv thov, yog li peb txiav txim lub hnub tiv thaiv yam tseem ceeb (SP) ntawm peb cov extracts. SPF qhia txog lub peev xwm ntawm ib yam khoom los nqus UV rays, tiv thaiv daim tawv nqaij los ntawm cov tshuaj lom uas tsim los ntawm xws li hluav taws xob. Cov tshuaj pleev ib ce cog qoob loo muaj peev xwm nqus tau UV hluav taws xob vim tias cov nroj tsuag rho tawm muaj polyphenols, xws li flavonoids lossis carotenoids. Cov tebchaw no, muaj cov nplhaib uas muaj ntxhiab, tuaj yeem nqus tau UV rays thiab, yog li ntawd, tuaj yeem ua lub hnub lim. Txij li thaum cov txiv hmab txiv ntoo extracts ntawm W.filifera muaj phenolic thiab flavonoid tebchaw [7], cov duab tiv thaiv los ntawm cov extracts tau raug soj ntsuam. Raws li pom nyob rau hauv Table 3, tag nrho cov kev soj ntsuam cov extracts, nyob rau hauv lub concentration ntawm 100 ug / mL, pom SPF qhov tseem ceeb ntawm 1.52 txog 3.35. Methanol extracts tau qhia kom muaj cov duab tiv thaiv zoo tshaj plaws. UV rays yog lub luag hauj lwm rau cov kab mob ntawm daim tawv nqaij thiab lawv ua rau cov txheej txheem uas ua rau cov tawv nqaij laus, oxidative kev nyuaj siab, thiab wrinkle tsim. Yog li, txo qhov nqus ntawm cov hluav taws xob no txhim kho, nyob rau hauv txoj kev tsis ncaj ncees, kev ua haujlwm antioxidant thiab inhibition ntawm aging-txog enzymes.

2.4. Cell Viability thiab Intracellular ROS Level

Txij li thaum oxidative kev nyuaj siab yog qhov tseem ceeb hauv kev ua rau kev laus thiab kev puas tsuaj rau lub hnub nyoog, peb kuj tau tshuaj xyuas seb W. filifera extracts inhibited HO2-induced ROS tiam nyob rau hauv lub cellular system. Hauv daim ntawv dhau los, peb tau piav qhia txog cov haujlwm antioxidant ntawm cov noob rho tawm siv txoj kev spectrophotometric (ABTS assay)[7]. Cov qauv tau qhia tias yog qhov zoo ntawm cov phenolic tebchaw nrog cov khoom antioxidant, nrog MEG qhia txog kev ua haujlwm zoo tshaj plaws. Txij li thaum cov extract no muaj qhov zoo tshaj plaws antioxidant kev ua si thiab kuj muaj peev xwm zoo, xav txog cov kev ua ub no tiv thaiv cov enzyme kuaj (cholinesterase, xanthine oxidase, thiab cov laus enzymes qhia ntawm no), peb txiav txim siab kom paub meej tias cov antioxidant muaj peev xwm ntawm MEG nyob rau hauv lub cellular qauv.

Ua ntej, cov teebmeem ntawm W.filifera extract ntawm cell viability raug tshawb xyuas hauv HaCaT hlwb. Lub immortalized tib neeg keratinocytes HaCaT cell kab tau siv dav los ua tus qauv los kawm txog epidermal homeostasis [15]. Txhawm rau txiav txim siab txog kev nyab xeeb ntawm cov extract no, cov hlwb raug kho nrog ntau qhov ntau ntawm cov qauv rau 24 teev thiab tshuaj xyuas siv MTT xeem. Cov txiaj ntsig tau qhia tias cov extract tsis yog cytotoxic hauv HaCaT hlwb thiab tsuas yog txo qis me me (kev muaj peev xwm ntawm 80 feem pua) tau pom ntawm 100 ug / mL (Daim duab 4). Txij li thaum lub viability tsis cuam tshuam txog 50 ug / mL (kev muaj peev xwm ntawm 96 feem pua), peb tau txiav txim siab ua qhov kev sim ntxiv ntawm tes siv txog qhov kev tawm dag zog no. Peb ntsuas ROS qib hauv cov hlwb ua ntej thiab tom qab oxidative kev nyuaj siab, thiab tom qab kho nrog MEG. Txoj kev tshawb no tau ua los ntawm 21,7'-dichlorofluorescein diacetate (DCFH-DA), uas yooj yim diffuses los ntawm cell membrane thiab yog hydrolyzed los ntawm endogenous esterases rau DCFH.Rapid nce nyob rau hauv DCF qhia oxidation ntawm DCFH los ntawm intracellular ROS, xws li H2O2.Raws li pom nyob rau hauv daim duab 5, H2O2 incubation ho nce ROS tsim nyob rau hauv HaCaT hlwb, tab sis kev kho nrog cov extract muaj peev xwm inhibit H2O2-induced ROS ntau lawm nyob rau hauv ib koob tshuaj teb. Yog li, cov txiaj ntsig no tau lees paub cov tshuaj tiv thaiv antioxidant thiab qhia tias MEG kuj tseem tuaj yeem txo qhov tsim ntawm ROS hauv cov hlwb.

Cov tshuaj methanolic extract tau txheeb xyuas hauv daim ntawv no tau pom tias muaj cov tshuaj antioxidant siab. Cov phenolic muaj pes tsawg leeg ntawm cov extract no muaj flavan-3-ol, thiab B-hom procyanidins yog lub ntsiab phenolic compounds [7]. Procyanidins, ib pawg ntawm polyphenolic bioflavonoids, tau tshaj tawm los nthuav tawm ntau yam kev lom neeg, tshuaj, thiab tshuaj tua kab mob tiv thaiv cov pa dawb radicals[16,17]. Ib txoj kev tshawb fawb yav dhau los tau qhia tias kev ua haujlwm ntawm cov tshuaj tiv thaiv radical ntawm procyanidins muaj zog ntawm cov concentrations siab [18]. Ntxiv mus, proanthocyanidin extracts yog cov zoo superoxide radical-scaveners tshaj antioxidant vitamin C thiab Trolox [19].

3. Cov ntaub ntawv thiab cov txheej txheem

3.1.Chemical

Tag nrho cov tshuaj reagents tau txais los ua cov khoom lag luam ntshiab los ntawm Sigma Chemical Co. (St. Louis, MO, USA) tshwj tsis yog qhia lwm yam, thiab siv yam tsis muaj kev lim dej ntxiv.

3.2.Kev npaj ua qauv cog

Cov txiv hmab txiv ntoo ntawm W.filifera tau sau hauv Tunisia hauv thaj chaw ntawm Gabes (G) thiab Sousse (S), thiab cov khoom siv cog tau npaj raws li cov txheej txheem yav dhau los tau piav qhia [7]. Pulp thiab noob tau cais lyophilized thiab tom qab ntawd cov ntaub ntawv cog (25 g) tau muab rho tawm hauv 100 mL dej (AE, aqueous extract), ethanol (E, ethanol extract), lossis methanol (ME, methanol extract) rau 72 h, ntawm chav tsev. kub, nyob rau hauv tas li stirring. Tom qab pom thiab centrifugation ntawm 10, 000 rpm, aqueous extracts ces lyophilized, thaum cov ethanol thiab methanol extracts tau concentrated nyob rau hauv lub nqus tsev vacuum, siv ib tug teb evaporator rau kev soj ntsuam ntxiv. Lub zog qhuav (1 mg / mL) tau yaj hauv DMSO ua ntej siv.

3.3.Enzymatic Inhibition

Cov txiaj ntsig ntawm txhua qhov kev ntsuam xyuas tau piav qhia hauv qab no tau qhia tias yog feem pua ​​​​ntawm kev tswj tsis pub muaj. Concentrations ntawm extracts uas ua rau 50 feem pua ​​​​ inhibition ntawm enzyme kev ua si (ICso) tau txiav txim los ntawm interpolation ntawm koob tshuaj teb curves. Tus qauv inhibition tau txiav txim siab los ntawm kev soj ntsuam ntawm qhov sib txawv ntawm qhov sib txawv ntawm cov substrate thiab qhov tsis muaj thiab muaj cov khoom rho tawm ntawm qhov sib txawv conceentrations. Cov ntaub ntawv Kinetics tau txheeb xyuas siv Lineweaver-Burk zajlus. Cov ntaub ntawv los ntawm kev soj ntsuam kev ua haujlwm tau raug kaw nrog Ultrospec 2100 spectrophotometer (Biochrom Ltd., Cambridge, UK).

3.3.1. Tyrosinase Inhibition Assay

Qhov inhibition ntawm tyrosinase kev ua los ntawm W.filifera extracts tau txiav txim los ntawm kev siv 3, 4-dihydroxyphenylalanine (L-DOPA) ua substrate [20]. Cov tshuaj tiv thaiv sib xyaw muaj 25 mM phosphate buffer (pH 6.8), nceb tyrosinase (100 U / mL, qhov kawg concentration), nrog los yog tsis muaj cov nroj tsuag extract tov. Tom qab ntawd, L-DOPA (0.5 mM) tau ntxiv rau hauv qhov sib tov thiab qhov kev ua haujlwm tau txiav txim siab los ntawm kev ua raws li qhov nce hauv absorbance ntawm 492 nm, uas tshwm sim los ntawm kev tsim cov khoom dopachrome. Qhov ntau ntawm cov extract siv rau nceb tyrosinase inhibition assay yog 0-0.3 mg/mL. Nyob rau hauv cov kev ntsuam xyuas ua tsis tau cov nroj tsuag rho tawm, DMSO tau ntxiv rau cov tshuaj tiv thaiv sib tov raws li ib tug dawb paug tswj. Kojic acid tau siv los ua kev tswj xyuas zoo.

3.3.2.Elastase Inhibition Assay

Elastase inhibition tau soj ntsuam los ntawm kev saib xyuas qhov tso tawm ntawm p-nitroaniline thaum lub hnub nyoog cleav-age ntawm substrate N-succ-(Ala)3-nitroanilide (SANA) los ntawm kev ua ntawm cov enzyme los ntawm txoj kev piav qhia [21], nrog rau kev hloov kho me ntsis. Qhov kev ntsuam xyuas tau ua tiav hauv 0.1 M Tris-HCl tsis nyob (pH8.{10}}). Porcine pancreatic elastase (3.3 ug / mL) tau incubated nrog los yog tsis muaj cov extract rau 20 min thiab, tom qab inculation, lub substrate (1.6mM) tau ntxiv, thiab cov enzyme kev ua ntawm 410 nm. Kev tswj hwm tau ua nrog DMSO, thaum oleanolic acid tau siv los ua kev tswj hwm zoo.

3.3.3.Collagenase Inhibition Assay

Collagenase los ntawm Clostridium histolyticum tau npaj rau hauv Tricine buffer {{{0}}}.05 M, pH 7.5, muaj 0.4 M NaCl thiab 0.01 M CaClz, thiab incubated (1 U / mL) nrog cov qauv kuaj ntawm qhov sib txawv ntawm qhov sib txawv rau 15 feeb. Cov hluavtaws substrate N-(3-[2-Fury]]-acryloyl)-Leu-Gly-Pro-Ala (FALGPA), npaj rau hauv tib txoj kev daws teeb meem, tom qab ntawd ntxiv los pib cov tshuaj tiv thaiv ( nrog rau qhov kawg concentration ntawm 0.8 mM).Kev nqus tau raug saib xyuas ntawm 340 nm [21]. Kev tswj hwm tau ua nrog DMSO, thaum epigallocatechin gallate tau siv los ua kev tswj hwm zoo.

3.4. Kev txiav txim siab ntawm In Vitro Sun Protection Factor

Lub hnub tiv thaiv qhov tseem ceeb ntawm W. filifera extracts tau txiav txim siab siv txoj kev UV ab-sorbance, raws li txoj kev piav qhia los ntawm Mansur li al. (1986) [22]. Cov absorbances ntawm cov extracts (0.1 mg/mL) tau ntsuas nyob rau hauv qhov ntau ntawm 290-320 nm, nrog 5 nm increments, thiab peb txiav txim siab tau ua nyob rau hauv txhua lub ntsiab lus. Tus SPF tau xam los ntawm kev siv Mansur Equation: qhov twg CF= kho qhov zoo (10); EE(A)= erythemogenic nyhuv ntawm hluav taws xob nrog nthwv dej-ntev λ;I()= hnub ci siv spectrum; Abs (λ= spectrophotometric absorbance qhov tseem ceeb ntawm wavelength λ. Tus nqi ntawm EE (A) × I (A) yog qhov tsis tu ncua. Lawv tau txiav txim los ntawm Sayre et al. (1979)[23] thiab muaj nyob rau hauv Table 4.

3.5.Cell Culture thiab Intracellular ROS Qib

Tib neeg cov tawv nqaij keratinocyte cell kab HaCaT yog kab lis kev cai hauv Dulbecco's Modified Eagle's Medium (DMEM) muaj 10 feem pua ​​​​fetal bovine serum (FBS, Gibco, NY, USA) thiab 1 feem pua ​​peni-cillin / streptomycin ntawm 37 degree, nyob rau hauv ib qho chaw humidified, nrog 5. feem pua ​​CO2.Cell viability tau kuaj pom los ntawm colorimetric 3-(4)5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay, as yav dhau los piav, nrog kev hloov kho me me [24]. Tom qab 24 teev incubation nrog MEG ntawm ntau qhov sib txawv (0-100 ug/mL), cov hlwb tau sau nrog MTT tov rau 3 teev ntawm 37 degree. Qhov tshwm sim violet formazan precipitates tau yaj hauv DMSO thiab qhov nqus ntawm txhua qhov dej tau txiav txim siab ntawm 560 nm siv tus nyeem ntawv microplate nrog 630 nm siv.

Cov qib cellular ROS tau txiav txim siab nrog txoj kev DCFH-DA [25]. HaCaT hlwb raug kho nrog ntau qhov ntau ntawm MEG (0-50 ug/mL) rau 24 teev. Tom qab ntawd, cov hlwb raug incubated nrog DCFH-DA (10 uM) ntawm 37 degree rau 30 min. Tom qab incubation, 1 mM HzO2 tau ntxiv rau hauv qhov dej, thiab qhov fluorescence siv ntawm DCF tau ntsuas tam sim ntawd siv lub fluorescent phaj nyeem ntawv ntawm qhov excitation wavelength ntawm 485 nm thiab emission wavelength ntawm 530 nm, nyeem ntawv ntawm 5 min rau 50. min.

3.6.Data Analysis

Tag nrho cov kev sim tau ua nyob rau hauv triplicates thiab cov ntaub ntawv tau qhia raws li qhov txhais tau tias 士 tus qauv sib txawv (SD). Kev sib piv ntawm cov pab pawg tau ua los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) ua raws li Tukey Multiple Comparisons Test. Ap-tus nqi tsawg dua 0.05 tau suav tias yog qhov tseem ceeb.

4. Cov lus xaus

Hauv kev xaus, peb tshaj tawm, thawj zaug, cov noob rho tawm los ntawm W.filifera muaj txiaj ntsig zoo hauv inhibiting cov enzymes tseem ceeb cuam tshuam rau ntawm daim tawv nqaij laus. Ntuj los yog "intrinsic" aging yog ib tug physiological phenomenon uas tshwm sim nyob rau hauv tag nrho cov tib neeg cov ntaub so ntswg raws li ib tug tshwm sim ntawm lub sij hawm. Txawm li cas los xij, daim tawv nqaij kuj raug rau sab nraud kev ntxhov siab-inducing yam uas sawv cev rau ib qho tseem ceeb ua rau cov tawv nqaij laus ntxov ntxov. "Extrinsic" kev laus yog feem ntau cuam tshuam nrog UV-induced kev puas tsuaj ntawm cov ntaub so ntswg ntawm daim tawv nqaij. UV rays ua rau oxidative kev nyuaj siab, uas yog lub luag hauj lwm rau lub activation ntawm lub enzymes degrading lub ECM, thiab cov tsos ntawm wrinkles thiab hnub nyoog me ntsis. Cov ntawv sau no qhia txog qhov tseem ceeb uas cov noob ntawm W.filifera tuaj yeem muaj nyob rau hauv cov ntsiab lus no. Raws li pom nyob rau hauv daim duab 6, cov extracts tuaj yeem ua rau kev tiv thaiv kev laus ntxov ntxov, ua haujlwm ib txhij ntawm ob peb sab.

Ua ntej, cov extracts tuaj yeem ua thaum pib ntawm cov txheej txheem los ntawm lawv cov teebmeem photoprotective thiab, yog li ntawd, txo UV ray absorption. Tom qab ntawd, lawv tau pom cov nyhuv antioxidant zoo nrog methanol extract, tiv thaiv ROS tsim nyob rau hauv lub cellular system, tsis muaj cell toxicity. Thaum kawg, tag nrho cov kev rho tawm, tshwj xeeb, cov qauv methanolic, tuaj yeem cuam tshuam collagenase, elastase, thiab tyrosinase (tom kawg mus rau ib qho me me). Zuag qhia tag nrho, cov txiaj ntsig tau pom tau tias W.filifera tuaj yeem yog qhov chaw ntawm bioactive molecules thiab txhawb kom muaj kev sim ntxiv txhawm rau txhawm rau cais cov khoom siv tib lub luag haujlwm rau cov haujlwm tau pom.

Kab lus no yog muab rho tawm los ntawm Nroj Tsuag 2021, 10, 151. https://doi.org/10.3390/plants10010151 https://www.mdpi.com/journal/plants