Hauv Vitro Kev Ntsuam Xyuas Ntawm Cov Photoreactivity Thiab Phototoxicity Ntawm Ntuj Polyphenol Antioxidants
Feb 21, 2022
Thov hu rauoscar.xiao@wecistanche.comkom paub ntau ntxiv.
Abstract:Polyphenols yog ib tsev neeg loj ntawm cov tebchaw ntuj siv dav hauv cov khoom siv tshuaj pleev ib ce vim lawv cov tshuaj tiv thaiv kab mob antioxidant thiab tiv thaiv kev mob nkeeg thiab lawv lub peev xwm los tiv thaiv UV hluav taws xob vim oxidative kev nyuaj siab. Txij li cov tebchaw no muaj chromophores thiab siv ncaj qha rau ntawm daim tawv nqaij, lawv tuaj yeem hnov mob nrog lub hnub ci thiab siv cov teebmeem phototoxic. Cov ntaub ntawv tshawb fawb muaj nyob rau ntawm lub peev xwm phototoxic ntawm cov tebchaw ntuj no tsis tshua muaj, thiab yog li lub hom phiaj ntawm txoj kev tshawb no yog los ntsuas qhov photoreactivity thiab phototoxicity ntawm tsib phenolic.antioxidantsnrog cov ntaub ntawv siv hauv cov khoom siv tshuaj pleev ib ce. Kev soj ntsuam ROS tus qauv tau raug lees paub thiab siv los tshuaj xyuas qhov photoreactivity ntawm natural phenolic antioxidants caffeic acid, ferulic acid, p-coumaric acid, 3, 4-dihydroxyphenylacetic acid (DOPAC), thiab rutin. Lub peev xwm phototoxicity tau txiav txim los ntawm kev siv tib neeg keratinocyte cell kab (HaCaT), raws li 3T3 Neutral Red Uptake phototoxicity test. Txawm tias tag nrhokawm phenolic antioxidantsabsorbed UV / Vis hluav taws xob nyob rau hauv thaj tsam ntawm 290 txog 700 nm, tsuas yog DOPAC tuaj yeem tsim cov pa oxygen. Lub cim ntawm reactive oxygen hom yog cov tshuaj tiv thaiv thaum ntxov raws li ib feem ntawm phototoxicity mechanism. Txawm li cas los xij, tsis muaj ib qho ntawm cov kev tshawb fawb txuas ntxiv txo qhov kev muaj peev xwm ntawm keratinocytes tom qab irradiation, ua rau kev txiav txim siab tias lawv tsis muaj peev xwm phototoxic. Cov ntaub ntawv tau txais nrog txoj haujlwm no qhia tias cov khoom sib txuas no muaj kev nyab xeeb thaum koom nrog hauv cov khoom siv tshuaj pleev ib ce.
Ntsiab lus:yees duab kev nyab xeeb; photoreactivity; phototoxicity; polyphenols;natural phenolic antioxidants; reactive oxygen hom; keratinocytes; kho tawv nqaij; cov khoom siv tshuaj pleev ib ce
Thov nias ntawm no kom paub ntxiv
Taw qhia
Polyphenols (PPs) yog ib qho ntawm feem ntau thiab dav faib pawg ntawm cov khoom ntuj tsim nyob rau hauv lub nceeg vaj cog. Cov qauv tshuaj ntawm PPs yog tus cwj pwm los ntawm muaj ib lossis ntau pawg phenolic hydroxyl, khi rau ib lossis ntau lub nplhaib benzene [1]. Polyphenols nthuav tawm ntau yam kev ua haujlwm lom neeg muaj txiaj ntsig zoo rau tib neeg, suav nrog tshuaj tua kab mob, tshuaj tua kab mob, tshuaj tua kab mob, tshuaj tiv thaiv kab mob, tshuaj tiv thaiv kab mob, thiab tshuaj tua kab mob antioxidant [2–5].Raws li antioxidants, polyphenols yuav tiv thaiv cell constituents tiv thaiv oxidative puas los ntawm reactive oxygen hom (ROS), xws li singlet oxygen, superoxide, thiab hydroxyl radicals, txwv tsis pub muaj kev pheej hmoo ntawm ntau yam kab mob txuam nrog oxidative kev nyuaj siab [6,7]. Vim lawv cov khoom siv tshuaj lom neeg, PPs muaj peev xwm rub tawm ROS thiab chelate hloov pauv hlau ions, xws li hlau thiab tooj liab, uas tau nyiam qhov kev nyiam ntawm kev lag luam tshuaj pleev ib ce rau lawv siv hauv kev tu tawv nqaij formulations [8-10]. Kev raug ultraviolet (UV) yog ib qho tseem ceeb ua rau mob qog noj ntshav ntawm daim tawv nqaij, thiab cov qog nqaij hlav tuaj yeem raug puas tsuaj ncaj qha los ntawm UV hluav taws xob lossis tsis ncaj qha los ntawm UV-mediated ROS overproduction [11]. Kev sim thiab kev tshawb fawb txog kab mob kis tau pom tias polyphenols tiv thaiv daim tawv nqaij los ntawm kev tsis zoo ntawm UV hluav taws xob los ntawm ntau txoj hauv kev [12]. Yog li ntawd, ob peb lub tebchaw ntawm tsev neeg no twb tau siv los ua cov khoom xyaw hauv ntau cov khoom lag luam kom zoo nkauj muaj nyob hauv khw [13]. Cov neeg siv khoom xav tau ntau ntxiv rau cov tshuaj pleev ib ce ntuj tau hais kom kev lag luam tsim cov qauv siv cov khoom siv ntuj tsim nrog cov khoom xyaw nquag, xws li polyphenols, uas yog ib qho kev cog lus thiab zoo rau kev tu tawv nqaij [13]. Txawm li cas los xij, cov chromophores tam sim no nyob rau hauv polyphenols cov qauv muaj peev xwm nqus tau UV / Vis hluav taws xob thiab raug tshuaj lom neeg ua rau muaj kev cuam tshuam ntawm cov xwm txheej uas yuav ua rau muaj kev cuam tshuam phototoxic [14]. Phototoxicity yog txhais raws li cov lus teb tshuaj lom los ntawm cov tshuaj pleev los yog cov tshuaj siv tshuaj photoreactive tom qab raug lub cev rau ib puag ncig lub teeb [15]. Cov khoom xyaw tshuaj nquag thiab cov khoom ntxiv rau kev tswj hwm lub cev, cov tshuaj kho mob rau cov tshuaj pleev ib ce, daim tawv nqaij daim tawv nqaij, thiab lwm yam, muaj peev xwm phototoxic thiab tuaj yeem ua rau muaj txiaj ntsig zoo rau cov tshuaj tiv thaiv phototoxic. Yog li ntawd, cov koom haum tswj hwm, US FDA, EU EMA, thiab ICH, muab cov lus qhia txog kev nyab xeeb ntawm cov duab, qhia txog cov kev sim thiab cov tswv yim ntsuas [15–17]. Kev ntsuas kev nyab xeeb ntawm cov khoom siv tshuaj pleev ib ce yog qhov yuav tsum tau ua raws li European Union txoj cai [18]. Qhov kev ntsuam xyuas kev nyab xeeb uas yuav tsum muaj suav nrog cov kev tshawb fawb txog tshuaj lom neeg ntawm cov khoom xyaw tshuaj pleev ib ce, suav nrog kev ntsuam xyuas cov duab toxicity [18]. Txij li thaum kuaj tsiaj tau raug txwv rau cov khoom siv tshuaj pleev ib ce, ntau qhov kev ntsuam xyuas kev nyab xeeb hauv vitro tau tshaj tawm xyoo dhau los, suav nrog UV spectrum tsom xam, 3T3 Neutral Red Uptake phototoxicity test (3T3 NRU PT), thiab cov pa oxygen reactive ( ROS) assay [18–20]. Kev soj ntsuam ROS tau tsim los rau kev ntsuam xyuas photoreactivity ntawm cov tshuaj, lub hauv paus ntsiab lus ntawm kev soj ntsuam cov tshuaj tiv thaiv photochemical hauv cov tshuaj ntsuam xyuas raug simulated hnub ci [19,20]. Los ntawm cov tshuaj tiv thaiv photochemical ROS, xws li superoxide anion thiab singlet oxygen, tuaj yeem tsim tawm, thiab cov txheej txheem photochemical tuaj yeem ua rau muaj kev cuam tshuam rau cov tshuaj tua kab mob phototoxicity [19,20]. Cov qib siab ntawm ROS tuaj yeem ua rau cytotoxicity los ntawm kev puas tsuaj ntawm DNA, lipids, thiab cov proteins los ntawm oxidative kev nyuaj siab.https://www.voachinese.com/a/us-lawmakers-united-condemn-russias-ukraine-invasion-20220224/6458599.htmlCov txheej txheem hauv vitro 3T3 NRU-PT siv cov nas fibroblasts cell kab Balb/c 3T3 thiab nruab nrab liab uptake ua qhov kawg ntawm cytotoxicity. Lub phototoxicity yog tom qab ntawd txiav txim siab los ntawm cov txheeb ze txo qhov kev muaj peev xwm ntawm cov hlwb raug rau cov tshuaj ntsuam xyuas nyob rau hauv lub xub ntiag thiab tsis muaj lub teeb simulating lub hnub ci. Cov kev tshawb fawb tau tshaj tawm hauv cov ntaub ntawv tau txiav txim siab tias qhov kev sim no dhau-rhiab heev, ua yuam kev kwv yees txog tsiaj thiab tib neeg kev nyab xeeb kev nyab xeeb, ua rau muaj ntau qhov tsis zoo thaum piv rau hauv vivo cov txiaj ntsig [21–23]. Xav txog qhov kev txwv no, kev hloov kho ntawm 3T3 NRU-PT txheej txheem tau npaj los ntawm peb pawg raws li kev siv tib neeg keratinocyte cell kab (HaCaT) [24]. Txij li cov txheej txheem no siv tib neeg cov keratinocyte hlwb, nws sawv cev rau tus qauv muaj tseeb dua, vim tias cov no yog hom cell uas muaj ntau tshaj plaws nyob rau hauv txheej txheej ntawm daim tawv nqaij, qhov twg cov tshuaj pleev ib ce tau siv thiab raug tshav ntuj [24]. Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog los kwv yees lub yees duab-induced toxicity peev xwm ntawm lub ntuj polyphenols p-coumaric acid, caffeic acid, 3, 4-dihydroxyphenylacetic acid (DOPAC), ferulic acid, thiab rutin (Daim duab 1), uas twb tau siv los ua cov khoom xyaw kom zoo nkauj lossis raug txiav txim siab vim lawv cov tshuaj lom neeg lom zem thiab antioxidant zog [25–27]. Kev soj ntsuam ROS tau raug lees paub thiab siv rau kev soj ntsuam cov duab kev nyab xeeb ntawm cov khoom sib txuas, thiab lawv cov cytotoxicity thiab phototoxicity tau soj ntsuam ntxiv siv tib neeg keratinocyte cell kab (HaCaT).
2. Cov txiaj ntsig thiab kev sib tham
Thawj qhov kev txiav txim siab rau qhov kev ntsuam xyuas ntawm qhov muaj peev xwm photoreactive yog seb cov khoom puas absorbs photons ntawm ib lub wavelength ntawm 290 thiab 700 nm. Ib qho chaw uas muaj molar extinction coefficient (MEC) ntau dua 1000 L mol−1 cm−1 ntawm ib qho wavelength ntawm 290 thiab 700 nm yog suav tias yog photoreactive txaus los ua rau phototoxicity ncaj qha [15]. Lub absorption spectra ntawm caffeic, p-coumaric, thiab ferulic acids, DOPAC, thiab rutin nyob rau hauv DMSO ntawm UV-pom lub teeb yog qhia nyob rau hauv daim duab 2. Tag nrho cov tebchaw absorbs nyob rau hauv lub spectral ntau ntawm 200 mus rau 700 nm, nrog siab tshaj wavelength ntawm los yog siab tshaj 290 nm thiab nrog MEC feem ntau ntau dua 4000 L mol−1 cm−1 (Table 1). Polyphenols yog cov khoom siv roj ntsha uas muaj π conjugated systems nrog phenolic hydroxyl pawg. π hom molecular orbitals 'kev hloov pauv hluav taws xob yog lub luag haujlwm rau UV-pom pom spectrum ntawm pawg neeg sib txuas no [28]
Tag nrho cov tshuaj ntsuam xyuas tau nthuav tawm MEC siab dua 1000 L mol−1 cm−1, uas txhais tau hais tias tag nrho cov muaj peev xwm phototoxic compounds tsim nyog kawm [15]. Ua ntej ua qhov kev ntsuam xyuas ROS, lub hnub ci simulator tau soj ntsuam, thiab cov kev sim ua kom zoo los xyuas kom meej tias qhov ntsuas qhov tseem ceeb ntawm singlet oxygen (SO) thiab superoxide anion (SA) nyob ze rau cov uas tau hais hauv cov ntaub ntawv [29]. Kev ua kom zoo ntawm ROS tiam kev soj ntsuam tau ua tiav siv cov kev tswj xyuas zoo thiab tsis zoo thiab kev tshawb fawb txog qhov ua tau tau ua los ntawm kev siv tshuaj siv tshuaj. Raws li kev sim siv, txhua yam khoom siv hauv qhov kev tshawb fawb muaj peev xwm ua tau raws li cov qauv kev lees paub muab qhov tseem ceeb rau SO thiab SA nyob rau hauv cov nqi nkag [29].
Kev ntsuam xyuas ROS tau ua tiav rau cov tshuaj polyphenolic, thiab lub peev xwm ntawm cov tshuaj ntsuam xyuas los tsim ROS, ntawm qhov concentration ntawm 200 µM, yog qhia nyob rau hauv Table 2. Table 2. Cov txiaj ntsig tau txais rau cov tshuaj ntsuam xyuas siv ROS assay.
Cov txiaj ntsig tau pom tau hais tias, tshwj tsis yog DOPAC, txhua qhov sib xyaw ua ke, tuaj yeem muab cais ua tsis yog photoreactive. Txawm hais tias cov tshuaj no tau pom lub teeb pom kev UV-pom pom thiab MEC siab dua 1000 L mol−1 cm−1 lawv tsis tau tsim ROS raws li kev sim, xws li SO lossis SA hom. Kuj ceeb tias, DOPAC muaj peev xwm ntxias cov tiam ntawm SO hom thiab, yog li ntawd, tau muab faib ua photoreactive, txawm tias qhov sib xyaw no muaj qhov qis tshaj MEC tus nqi hauv UV-pom ntau ntawm tag nrho cov tebchaw kawm. Yuav tsum muaj kev tshawb fawb ntxiv kom nkag siab txog cov txiaj ntsig tau txais rau DOPAC, uas kuj tseem yog qhov tseem ceeb, nyob rau yav tom ntej, los tsim kom muaj kev sib raug zoo ntawm cov qauv tshuaj ntawm ib qho chaw thiab nws lub peev xwm los ua photoreactive. Lub cytotoxicity ntawm DMSO siv rau kev soj ntsuam ntawm cov teebmeem phototoxic, nyob rau hauv lub xub ntiag thiab tsis muaj irradiation, tom qab 1h ntawm raug raug soj ntsuam. Rau cov phaj tsis-irradiated, DMSO tsis tau qhia qhov sib txawv tseem ceeb txheeb ze rau cov kev tswj tsis zoo. Txawm li cas los xij, rau cov phaj irradiated, muaj qhov sib txawv tseem ceeb ntawm 1 feem pua DMSO thiab cov kuab tshuaj tswj kev txheeb ze rau cov kev tswj tsis zoo (p <0.0001), uas="" ua="" rau="" kev="" siv="" cov="" kuab="" tshuaj="" tswj="" ntawm="" txhua="" qhov="" kev="" sim="" los="" lav="" tias="" qhov="" sib="" txawv="" ntawm="" cov="" cell="" viability.="" tau="" tsuas="" yog="" ntaus="" nqi="" rau="" cov="" tebchaw="" hauv="" kev="" kawm.="" txhawm="" rau="" kom="" ntseeg="" tau="" tias="" qhov="" ua="" tau="" zoo="" ntawm="" cov="" tshuaj="" tua="" kab="" mob="" phototoxicity="" siv="" tib="" neeg="" keratinocyte="" cell="" kab="" (hacat),="" 5-methoxy="" psoralen,="" chlorpromazine="" hydrochloride,="" thiab="" quinine="" raug="" kuaj="" raws="" li="" kev="" tswj="" zoo,="" thiab="" acetylsalicylic="" acid,="" hexachlorophene,="" thiab="" sodium="" lauryl="" sulfate="" raws="" li="" kev="" tswj="" tsis="" zoo.="" [24].="" cov="" txiaj="" ntsig="" tau="" los="" ntawm="" kev="" soj="" ntsuam="" phototoxicity="" piv="" cov="" cell="" viability="" ntawm="" hacat="" hlwb,="" irradiated="" thiab="" non-irradiated,="" nyob="" rau="" hauv="" lub="" xub="" ntiag="" ntawm="" cov="" tshuaj="" ntsuam="" xyuas="" (poly)="" phenolic="" tebchaw="" yog="" depicted="" nyob="" rau="" hauv="" daim="" duab="" 3.="" qhov="" ntau="" ntawm="" cov="" concentrations="" ntawm="" cov="" tshuaj="" ntsuam="" xyuas="" nyob="" rau="" hauv="" lub="" xub="" ntiag.="" (irr="" plus="" )="" thiab="" qhov="" tsis="" muaj="" (irr−)="" ntawm="" lub="" teeb="" tau="" txiav="" txim="" siab="" nyob="" rau="" hauv="" ntau="" qhov="" kev="" tshawb="" nrhiav,="" xav="" txog="" qhov="" siab="" tshaj="" plaws="" ntawm="" 1000="" µm.="" ib="" tug="" geometric="" dilution="" series="" tau="" siv="" thiab="" hloov="" kho,="" thaum="" tsim="" nyog,="" raws="" li="" ib="" tug="" muaj="" nuj="" nqi="" ntawm="" concentration-="" teb="" nyob="" rau="" hauv="" lub="" xub="" ntiag="" thiab="" tsis="" muaj="" irradiation.="" tsis="" pub="" dhau="" qhov="" kev="" sim="" siab="" ntau="" yam="" (12.5;="" 31.25;="" 62.5;="" 125;="" 250;="" 500,="" thiab="" 1000="" µm)="" tsis="" muaj="" cov="" tshuaj="" ntsuam="" xyuas="" tau="" ua="" rau="" 50="" feem="" pua="" ntawm="" cov="" cell="" muaj="" peev="" xwm="" txo="" qis,="" yog="" li="" ntawd,="" nws="" tsis="" tuaj="" yeem="" suav="" cov="" nqi="" sib="" xws="" ic50="" thiab="" pif.="" .="" ntawm="" qhov="" tsis="" sib="" xws,="" nws="" muaj="" peev="" xwm="" pom="" tau="" tias="" qhov="" kev="" siv="" tshuaj="" ntau="" ntxiv="" hauv="" kev="" muaj="" peev="" xwm="" ntawm="" cov="" hlwb="" irradiated="" nyob="" rau="" hauv="" lub="" xub="" ntiag="" ntawm="" cov="" tshuaj="" ntsuam="" xyuas,="" tejzaum="" nws="" yog="" vim="" photoprotective="" los="" ntawm="" cov="" antioxidants="" tiv="" thaiv="" oxidative="" puas="" tsuaj="" los="" ntawm="" hluav="" taws="" xob="" ua="" rau="" ib="" tug="" ntau="" dua="" feem="" pua.="" ntawm="" cov="" hlwb="" siv="" tau="" thaum="" piv="" rau="" kev="" tswj="" hwm="" (tsis="" kho="" cov="" hlwb="" irradiated).="" nws="" tau="" piav="" nyob="" rau="" hauv="" cov="" ntaub="" ntawv="" hais="" tias="" uv="" hluav="" taws="" xob="" ua="" rau="" lub="" cim="" ntawm="" ros,="" ib="" tug="" overproduction="" ntawm="" nitric="" oxide,="" thiab="" depletion="" ntawm="" antioxidant="" tiv="" thaiv="" nyob="" rau="" hauv="" keratinocytes="" [30].="" rau="" cov="" laj="" thawj="" no,="" polyphenols="" nrog="" lub="" peev="" xwm="" antioxidant="" tau="" kawm="" ua="" cov="" tshuaj="" tiv="" thaiv="" photoprotective.="" txawm="" hais="" tias="" feem="" ntau="" cov="" kev="" tshawb="" fawb="" tsom="" mus="" rau="" lub="" peev="" xwm="" photoprotective="" ntawm="" polyphenols,="" lawv="" tsis="" tau="" kawm="" txog="" lawv="" cov="" peev="" xwm="" phototoxic.="" cov="" kev="" tshawb="" fawb="" yav="" dhau="" los="" tau="" lees="" paub="" qhov="" muaj="" peev="" xwm="" ntawm="" caffeic,="" ferulic,="" thiab="" p-coumaric="" acids="" kom="" tshem="" tawm="" ros="" thiab="" reactive="" nitrogen="" hom="" (rns).="" ntxiv="" mus,="" kev="" tiv="" thaiv="" tawm="" tsam="" cov="" teebmeem="" ntawm="" uv="" hluav="" taws="" xob="" kuj="" tau="" tsim="" nyob="" rau="" hauv="" vivo="" lossis="" tawv="" nqaij="" hlwb="" rau="" peb="" cov="" phenolic="" tebchaw="" [31–34].="" cov="" ntaub="" ntawv="" no="" yuav="" piav="" qhia="" txog="" qhov="" nce="" hauv="" feem="" pua="" ntawm="" cov="" hlwb="" siv="" tau="" thaum="" raug="" irradiation="" nyob="" rau="" hauv="" lub="" xub="" ntiag="" ntawm="" cov="" tebchaw="" nyob="" rau="" hauv="" kev="" kawm.="" rutin,="" zoo="" li="" caffeic,="" ferulic,="" thiab="" p-coumaric="" acids="" kuaj="" tsis="" zoo="" nyob="" rau="" hauv="" ros="" tiam="" kev="" soj="" ntsuam="" thiab="" tsis="" induce="" phototoxicity="" hauv="" hacat="" cell="" kab.="" muaj="" qee="" cov="" ntaub="" ntawv="" tsis="" sib="" xws="" hauv="" cov="" ntaub="" ntawv="" hais="" txog="" rutin's="" phototoxic="" peev="" xwm,="" yog="" li="" kev="" txaus="" siab="" ntawm="" cov="" txiaj="" ntsig="" tau="" txais.="" qhov="" kev="" ntsuam="" xyuas="" ntawm="" phototoxic="" muaj="" peev="" xwm="" siv="" lub="" keratinocyte="" cell="" system="" (hacat),="" pom="" tias="" rutin="" pom="" tau="" tias="" phototoxicity="" [35].="" ntawm="" qhov="" tsis="" sib="" xws,="" siv="" qhov="" kev="" sim="" teeb="" tsa="" uas="" ua="" haujlwm="" capillary="" electrophoresis="" nrog="" electrochemical="" thiab="" uv="" nrhiav="" kom="" pom="" cov="" duab="" toxicity="" ntawm="" cov="" nroj="" tsuag="" rho="" tawm="" thiab="" cov="" khoom="" hauv="" cov="" ntsiab="" lus="" ntawm="" oxygen="" noj="" thiab="" tsim="" cov="" pa="" oxygen="" reactive="" hom="" thaum="" irradiation="" nrog="" pom="" lub="" teeb,="" nws="" muaj="" peev="" xwm="" txiav="" txim="" siab="" tias="" rutin="" yog.="" tsis="" phototoxic="" [36],="" uas="" tau="" pom="" zoo="" nrog="" cov="" txiaj="" ntsig="" tau="" txais="" hauv="" txoj="" haujlwm="" no="" qhov="" twg="" rutin="" tsis="" qhia="" photoreactivity="" vim="" nws="" tsis="" tau="" tsim="" so="" lossis="" sa="" hauv="" ros="" tiam="" kev="" soj="" ntsuam.="" ntawm="" qhov="" tod="" tes,="" hauv="" txoj="" haujlwm="" no,="" nws="" kuj="" tau="" pom="" tias="" rutin="" los="" ntawm="" nws="" tus="" kheej="" nthuav="" tawm="" tsis="" muaj="" peev="" xwm="" phototoxic="" hauv="" hacat="" cell="" kab.="" cov="" kev="" tshawb="" pom="" tsis="" sib="" xws="" no="" qhia="" txog="" qhov="" tseem="" ceeb="" ntawm="" kev="" siv="" cov="" qauv="" ntsuas="" kev="" ntsuas="" thiab="" tseem="" siv="" lub="" teeb="" pom="" kev="" zoo="" kom="" tsis="" txhob="" muaj="" qhov="" yuam="" kev.="" interestingly,="" thiab="" txawm="" hais="" tias="" tus="" qauv="" zoo="" sib="" xws="" ntawm="" dopac="" thiab="" lwm="" yam="" pps="" tau="" kawm,="" dopac="" tsim="" so="" hauv="" ros="" tiam="" kev="" soj="" ntsuam,="" thiab="" tau="" muab="" cais="" ua="" photoreactive.="" txawm="" li="" cas="" los="" xij,="" thaum="" kuaj="" hauv="" hacat="" kab="" ntawm="" tes,="" dopac="" pom="" tias="" tsis="" yog-phototoxic.="" los="" ntawm="" cov="" txiaj="" ntsig="" tau="" txais,="" dopac="" zoo="" li="" photoreactive="" tab="" sis="" tsis="" yog="" phototoxic,="" yog="" li="" nws="" tsis="" xav="" tias="" cov="" tshuaj="" tiv="" thaiv="" phototoxic="" tshwm="" sim="" tom="" qab="" siv="" tshuaj="" pleev="" xim="" ntawm="" cov="" tshuaj="">
3. Cov ntaub ntawv thiab cov txheej txheem
3.1. Reagents
3, 4-Dihydroxyphenylacetic acid (DOPAC), caffeic acid, trans-ferulic acid, p-coumaric acid, rutin, chlorpromazine hydrochloride, disodium hydrogen phosphate dodecahydrate, sodium phosphate monobasic monohydrate, Neutral Red (NR), thiab dimethyl sulfoxide (DMSO) tau yuav los ntawm Sigma-Aldrich (Madrid, Spain). Quinine hydrochloride, benzocaine, diclofenac, thiab erythromycin tau yuav los ntawm Acofarma (Madrid, Spain). Lub immortalized human keratinocyte (HaCaT) cell kab tau txais los ntawm Cell Lines Service (CLS) (Eppelheim, Germany). Dulbecco's Modified Eagle Medium (DMEM) nrog 4.5 g / L D-glucose, L-glutamine, 25 mM HEPES, thiab DMEM nrog 4.5 g / L D-glucose, L glutamine, 25 mM HEPES tsis muaj phenol liab, Dulbecco's Phosphate Buffe (DPBS), Fetal Bovine Serum (FBS), thiab trypsin EDTA tau yuav los ntawm Gibco Life Technologies (Waltham, MA, USA). Ethanol tau muab los ntawm Aga (Lisbon, Portugal). N, N-Dimethyl{14}}nitroguanidine (RNO), imidazole, thiab Nitro Blue Tetrazolium (NBT) tau yuav los ntawm Alfa Aesar (Kandel, Lub teb chaws Yelemees).
3.2. Spectral Absorption
Kev nqus spectrum ntawm txhua qhov kev tshawb fawb tau txiav txim siab nyob rau hauv thaj tsam ntawm 290 txog 700 nm, raws li OECD Test Guideline 101, siv Jasco V650 UV / VIS spectrophotometer [37]. Cov tshuaj tau yaj hauv DMSO txhawm rau kom tau txais qhov kawg ntawm 10 µg / mL thiab qhov nqus spectra tau ntsuas siv UV pob tshab quartz cuvettes (txoj kab ntev=10 hli). Txhua lub spectrum raug kho kom haum rau cov kuab tshuaj tshwj xeeb hauv lub hauv paus nqus. Molar extinction coefficients (MEC) tau suav nrog qhov siab tshaj plaws nqus peaks ntawm 290 txog 700 nm [15].
3.3. Reactive Oxygen Species (ROS) Assay
ROS assay raws tu qauv tau tsim, thiab cov kev tshawb fawb validation tau ua raws li cov txheej txheem piav nyob rau hauv cov ntaub ntawv [19,29]. Cov kev daws teeb meem ntawm tag nrho cov tshuaj ntsuam xyuas tau npaj ntawm 10 mM concentration hauv DMSO thiab siv nyob rau hauv tib hnub, tiv thaiv los ntawm lub teeb. Luv luv, singlet oxygen (SO) tiam tau kuaj pom los ntawm kev ntsuas spectrophotometric ntawm p-nitrosodimethylaniline (RNO) bleaching ntawm 440 nm siv imidazole ua ib qho kev xaiv ntawm singlet oxygen. Cov qauv, uas muaj cov tshuaj ntsuam xyuas (200 µM), RNO (50 µM), thiab imidazole (50 µM) hauv 20 mM sodium phosphate buffer (PB, pH 7.4), tau muab tso rau hauv ib lub raj thiab tov nrog vortex tov thiab sonicated, tiv thaiv los ntawm lub teeb, rau 10 min. Cov sib tov tau raug xa mus rau hauv Hellma quartz iav ua haujlwm siab ntawm tes thiab kuaj xyuas nag lossis daus hauv lub tshuab ntsuas ua ntej lub teeb raug. Tom qab ntawd, cov qauv raug irradiated siv Fitoclima S600PL thermostatic solar simulator (Aralab, Portugal), nruab nrog yim Repti Glo (20 W) UV-Vis teeb, rau 90 min ntawm 25 ◦C. Tom qab irradiation, lub absorbance tau nyeem dua ntawm 440 nm. Superoxide anion (SA) tiam tau kuaj pom los ntawm kev soj ntsuam qhov txo qis ntawm nitroblue tetrazolium (NBT) rau monoformazan (NBT ntxiv), qhov tsim ntawm uas tuaj yeem soj ntsuam spectrophotometrically ntawm 560 nm. Cov qauv uas muaj cov tshuaj ntsuam xyuas (200 µM) thiab NBT (50 µM) hauv 20 mM NaPB tau irradiated, thiab qhov txo qis hauv NBT tau ntsuas los ntawm qhov nce hauv absorbance ntawm 560 nm tib yam li rau kev txiav txim siab SO. Cov kev sim tau ua nyob rau hauv triplicate. Txij li lub hnub ci simulator siv txawv ntawm cov qauv pom zoo, nws yog ib qho tsim nyog yuav tsum tau ua raws li cov txheej txheem irradiation. Kev ntsuam xyuas ROS tau ua tiav los xyuas kom meej tias cov kab mob irradiation txaus siab rau cov kev pom zoo uas siv cov tshuaj zoo (quinine) thiab kev tswj tsis zoo (sulisobenzone) thiab cov tshuaj siv tshuaj sib txuas [29]. Raws li qhov tshwm sim (txhais tau tias ntawm kev txiav txim siab triplicate) los ntawm ROS kev soj ntsuam, cov tshuaj polyphenolic kuaj tau muab cais ua cov tshuaj photoreactive thaum SO tus nqi 25 lossis ntau dua thiab / lossis SA tus nqi ntawm 20 lossis ntau dua raug ntsuas; nyob rau hauv lem, nws tau txiav txim tias yog ib yam khoom uas tsis yog-photoreactive thaum qhov tseem ceeb ntawm tsawg dua 25 rau SO thiab tsawg dua 20 rau SA tau sau tseg [29].
3.4. Cell Culture
Cov hlwb HaCaT tau khaws cia ntawm 37 ◦C nyob rau hauv ib qho chaw humidified ntawm 95 feem pua ntawm huab cua thiab 5 feem pua CO2 nyob rau hauv lub incubator hauv DMEM nrog 10 feem pua FBS thiab 1 feem pua cov tshuaj tua kab mob. Siv lub tshuab ntsuas inverted, lub cell confluence tau pom thiab yog tias cov hlwb mus txog 70-80 feem pua confluence, subculture tau ua los tiv thaiv cell tuag. Rau lub hom phiaj no, kab lis kev cai nruab nrab yog aspirated, thiab cov hlwb raug ntxuav nrog DPBS, 2 mL ntawm trypsin 0.25 feem pua yog ntxiv thiab incubated rau 7 mus rau 8 min ntawm 37 ◦C hauv 5 feem pua CO2 cua. Tom qab cov hlwb raug tshem tawm, qhov nruab nrab tshiab tau ntxiv los thaiv qhov kev ua haujlwm trypsin. Rau kev suav ntawm tes, 10 µL ntawm kev ncua ntawm tes tau muab tso rau hauv Neubauer chamber uas cov hlwb raug suav. Cov tau txais xov tooj raug tshem tawm ces muab faib mus rau hauv lub khob tshiab nrog rau cov kab lis kev cai tshiab ntawm tes. Rau kev khov ntawm tes, DMSO (5 feem pua v / v) tau siv los ua cryo-preservative los tiv thaiv kev tsim cov muaju thaum lub sijhawm khaws cia. Txhawm rau txiav txim siab lub sijhawm tsim nyog rau cov hlwb kom du, 1 × 106 hlwb tau cog rau hauv tsib 75 cm2 flasks thiab incubated rau 24 h ntawm 37 ◦C hauv 5 feem pua CO2 cua kom ncav cuag kev ua tiav. Tom qab ntawd, cov hlwb ntawm txhua lub raj mis raug suav rau lub sijhawm sib txawv. Cov txiaj ntsig tau raug teeb tsa hauv daim duab uas sawv cev rau tus lej xov tooj ntawm tes piv rau lub sijhawm, los ntawm qhov uas lub sijhawm ua ob npaug tau suav nrog siv kev txheeb xyuas kab rov tav. Lub sijhawm suav ob npaug tau txais yog 20.43 h, uas yog raws li cov txiaj ntsig tau tshaj tawm hauv cov ntaub ntawv, yog li lees paub tias cov hlwb siv tau nyob rau hauv ib txwm muaj kev loj hlob [38]. 3.5. Phototoxicity Assay Rau kev kawm txog phototoxicity, yav dhau los raws tu qauv siv hauv peb chav kuaj tau ua raws [24]. Qhov siab ntawm UVA / UVB Osram teeb (240V E27) tau hloov kho kom irradiate lub hlwb nrog irradiation UVA koob ntawm 1.7 mW / cm2 (Cosmedico radiometer, UVM-7), raws li OECD cov lus qhia [23] . Thaum lub sij hawm irradiation (10 min), cov phiaj tau khaws cia rau hauv ib tus neeg tau txais styrofoam uas muaj cov dej txias txias, thiab ntsuas qhov ntsuas kub thoob plaws hauv cov txheej txheem. Luv luv, los ua NR uptake assay, HaCaT hlwb tau noob (2 × 104 hlwb/zoo) thiab incubated ntawm 37 ◦C hauv 5 feem pua CO2 cua rau 24 teev. Tom qab ntawd, qhov nruab nrab raug tshem tawm, qhov sib txawv ntawm cov tshuaj ntsuam xyuas tau ntxiv, thiab cov hlwb raug tsim nyob rau hauv tib lub sijhawm rau 1 teev. Ib phaj tau khaws cia hauv qhov tsaus ntuj thaum lwm lub phaj tau irradiated rau 10 min nrog qhov kub thiab txias ntawm 29-32 ◦C. Tom qab ntawd, cov hlwb nruab nrab tau hloov nrog DMEM tshiab yam tsis muaj phenol liab thiab incubated rau 18-22 teev. Tom qab lub sij hawm incubation no, cov hlwb ntawm ob daim hlau raug ntxuav nrog DPBS thiab ua tiav DMEM uas muaj 50 µg / mL NR ntxiv rau txhua qhov dej thiab incubated rau 3 teev. Tom qab incubation nrog NR, NR tov tau muab tshem tawm, thiab ib tug NR desorb tov (50 feem pua ethanol: 1 feem pua acetic acid: 49 feem pua cov dej distilled) tau ntxiv rau rho tawm NR dye los ntawm cov hlwb. Rau kev nyeem ntawv, qhov nqus tau ntsuas ntawm 540 nm. Hauv txhua lub phaj, DMSO tswj tau raug kuaj. Cell viability cov ntaub ntawv tau los ntawm txhua lub phaj tau qhia raws li qhov sib piv ntawm absorbance ntawm cov hlwb kho rau cov kuab tshuaj tswj cov hlwb thiab tau siv ntxiv los kwv yees IC50 qhov tseem ceeb uas siv cov kev ntsuam xyuas kab rov tav. Tus nqi Photo-Irritation-Factor (PIF) tus nqi rau txhua yam tshuaj ntsuam xyuas tau xam raws li qhov sib piv ntawm IC50 tus nqi ntawm irradiated (Irr plus) piv rau IC50 tus nqi ntawm cov hlwb uas tsis yog-irradiated (Irr−). Raws li OECD cov lus qhia, PIF Performance index qis dua 2 kwv yees qhov tsis muaj cov nyhuv phototoxic, PIF Performance index ntawm 2 thiab 5 kwv yees qhov tshwm sim ntawm cov nyhuv phototoxic, thiab PIF siab dua 5 kwv yees cov nyhuv phototoxic [23]. Tested compounds raug soj ntsuam nyob rau hauv ib tug ntau yam ntawm cov concentrations: 12.5; 31.25; 62,5; 125; 250; 500 thiab 1000 µM. 3.6. Kev Tshawb Fawb Txog Kev Tshawb Fawb Tag nrho cov ntaub ntawv tau nthuav tawm raws li qhov txhais tau tias ± tus qauv sib txawv (SD) los ntawm tsawg kawg peb qhov kev sim ywj pheej khiav hauv triplicate. Txhawm rau kom paub meej qhov zoo ib yam thiab homogeneity ntawm qhov sib txawv, D'Agostino-Pearson omnibus normality test tau siv, thiab tom qab ntawd, ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA), ua raws li Dunnett's post hoc test (piv rau cov tswj tsis zoo nrog cov kuab tshuaj), tau ua. Cov duab tau tsim nrog software GraphPadPrism rau Windows (version 6.0, GraphPad Software, Inc. (San Diego, CA, USA).
4. Cov lus xaus
Nyob rau hauv txoj kev tshawb no, ib tug Reactive Oxygen Species (ROS) assay thiab 3T3 Neutral Red UptakePhototoxicity assay (3T3 NRU-PT) tau siv los tshuaj xyuas tus cwj pwm ntawm natural phenolic antioxidants thaum raug hluav taws xob ua rau lub hnub ci txhawm rau tshuaj xyuas lawv cov photoreactivity thiab phototoxicity. peev xwm. Cov txiaj ntsig tau tso cai qhov kev txiav txim siab tias, txawm hais tias rutin thiab caffeic, p coumaric, thiab ferulic acids nqus UV-pom lub teeb hluav taws xob thiab tam sim no MEC siab dua 1000 L mol-1cm-1, lawv raug cais raws li tsis yog-photoreactive. Ntxiv mus, cov tebchaw no tsis ua rau muaj kev phom sij thaum kuaj siv HaCaT cell kab. Cov ntaub ntawv pom tau hais tiascov antioxidants nolos ntawm lawv tus kheej tsis xav tias yuav ua rau phototoxicity thiab yog li lawv tuaj yeem suav tias muaj kev nyab xeeb rau kev siv hauv cov tshuaj pleev ib ce. Ntawm qhov tod tes, nws muaj peev xwm soj ntsuam qhov nce ntawm cov cell muaj peev xwm raug rau cov hluav taws xob thaum nyob rau hauv lub xub ntiag ntawm cov antioxidants nthuav tawm cov nyhuv photoprotective uas yuav txaus siab rau kev kawm los txhawb lawv txoj kev siv cov tshuaj photoprotective hauv cov tshuaj pleev ib ce. Nyob rau hauv rooj plaub ntawm DOPAC, qhov sib xyaw no tau pom tias yog photoreactive, txawm hais tias tsis muaj kev cuam tshuam phototoxicity hauv 3T3 Neutral Red Uptake assay. Txawm li cas los xij, kev tshawb fawb ntxiv yuav tsum tau ua kom nkag siab txog cov txheej txheem tom qab DOPAC photoreactivity, kom ntseeg tau tias nws cov duab muaj kev nyab xeeb, thiab tseem yuav nkag siab txog lub luag haujlwm thiab kev sib raug zoo ntawm cov qauv tshuaj thiab lub peev xwm ntawm cov khoom siv los ua photoreactive. Sau Kev Koom Tes: Kev Tshawb Fawb, BA; Kev Sau Ntawv—Thawj Draft Npaj, BA; Conceptualization IFA, HC, thiab JG: Resources, IFA, HC, JMSL, thiab JG; Kev Sau Ntawv—Saib xyuas & Hloov Kho, IFA, HC, JMSL, thiab JG; Kev saib xyuas, IFA, HC, thiab JG; Kev Tau Txais Nyiaj Txiag, IFA, HC, JMSL, thiab JG Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm phau ntawv sau.Funding: Qhov kev tshawb fawb no tsis tau txais nyiaj txiag sab nraud.Institutional Review Board Statement: Tsis muaj.Informed Consent Statement: Tsis siv.Data Daim Ntawv Qhia Txog Kev Muaj: Tag nrho cov ntaub ntawv uas tau nthuav tawm hauv txoj kev tshawb fawb no muaj nyob rau hauv tsab xov xwm.Kev lees paub: Txoj haujlwm no yog nyiaj txiag los ntawm lub teb chaws cov nyiaj los ntawm FCT-Fundação para a Ciência ea Tecnologia, IP, nyob rau hauv qhov project UIDP/04378/2020, UIDB /04378/2020 ntawm ResearchUnit ntawm Applied Molecular Biosciences-UCIBIO thiab qhov project LA/P/0140/2020 ntawm AssociateLaboratory Institute for Health and Bioeconomy-i4HB thiab UIDB/00081/2020 grant-funded by FCT/MCTES. Kev tsis sib haum xeeb ntawm kev txaus siab: Cov neeg sau ntawv tshaj tawm tsis muaj teeb meem ntawm kev txaus siab.Sample Muaj: Tsis siv tau.
Cov ntaub ntawv
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