Bioactive Bacterial Nanocellulose Membranes Enriched Nrog Eucalyptus Globulus Labill. Leaves Aqueous Extract Rau Anti-Aging Skin Care Applications

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

Abstract:Cov kab mob nanocellulose (BNC) daim nyias nyias, nrog rau lub cev thiab lub zog zoo kawg li, tau tshwm sim los ua ntau yam biopolymeric cab kuj ntawm bioactive compounds rau daim tawv nqaij daim ntaub ntawv. Hauv txoj kev tshawb no, BNCmembranes tau ntim nrog glycerol (raws li plasticizer thiab humectant tus neeg sawv cev) thiab ntau npaum li cas (1-3 ug cm-2) ntawm cov aqueous extract tau los ntawm hydro-distillation ntawm Eucalyptus globulus Labill. nplooj (HDE), rau daim ntawv thov ua daim ntawv npog ntsej muag. Txhua daim nyias nyias muaj peev xwm tiv taus thiab ua tau zoo heev hauv cov xeev qhuav thiab ntub dej, nrog cov khoom zoo sib xws lossis txawm tias zoo dua li cov khoom lag luam BNC daim npog ntsej muag. Tsis tas li ntawd, HDE tau pom los muab cov tshuaj tiv thaiv kab mob antioxidant rau BNC ntshiab. Tsis tas li ntawd, thaum 3 lub hlis ntawm kev khaws cia ntawm 22-25 degree thiab 52 feem pua ​​​​ntawm cov av noo (RH) lossis ntawm 40C thiab 75 feem pua ​​​​RH, nws tau lees paub tias cov tshuaj tiv thaiv antioxidant thiab macroscopic nam ntawm daim nyias nyias nrog 2 ug cm {{10 }} ntawm HDE tau khaws cia. Membranes kuj tau pom tias tsis yog-cytotoxic rau HaCaT thiab NIH / 3T3 hlwb, thiab daim nyias nyias nrog 2ug cm-2 ntawm HDE ua rau muaj kev txo qis hauv cov senescence-associated -galactosidase kev ua hauv NIH/3T3 hlwb. Cov kev tshawb pom no qhia txog qhov tsim nyog thiab muaj peev xwm ntawm daim nyias nyias tau ua bioactive lub ntsej muag lub ntsej muag rau kev siv tshuaj tiv thaiv kev laus.

Thov nias ntawm no kom paub ntxiv

Ntsiab lus:kab mob nanocellulose; Eucalyptus globulus Labill. nplooj; aqueous extract; kev ua haujlwm antioxidant; daim npog ntsej muag; anti-aging; daim ntawv thov kev tu tawv nqaij

1. Taw qhia

Cov pej xeem hauv ntiaj teb no muaj hnub nyoog loj heev vim qhov poob qis hauv kev yug me nyuam thiab kev txhim kho ntawm kev ciaj sia, thiab qhov nce hauv qhov nruab nrab lub neej expectancy, cuam tshuam nrog kev kho mob thiab kev tsim kho thev naus laus zis tau ua tiav nyob rau xyoo dhau los [1,2]. Lub ntiaj teb cov pej xeem hnub nyoog 60 los yog siab dua yuav tsum ncav cuag li 2.1 billion los ntawm 2050, sawv cev nce ntawm kwv yees li ob npaug los ntawm 2017, thiab tus naj npawb ntawm cov neeg muaj hnub nyoog 80 los yog tshaj yuav tsum muaj ze li peb npaug ntawm tib lub sijhawm 3]. Txawm li cas los xij, txawm tias kev txhim kho lub neej ntev, kev laus ntawm daim tawv nqaij yog ib qho txheej txheem inevitable, thiab yog li ntawd, kev xav tau ntau ntxiv rau cov khoom tu tawv nqaij los tiv thaiv kev laus.

Kev laus ntawm daim tawv nqaij yog cov txheej txheem lom neeg ua ke nrog cov txheej txheem endogenous thiab exogenous [4]. Endogenous aging yog tsuas yog txiav txim los ntawm caj ces yam thiab hormonal hloov uas tshwm sim nrog cov txheej txheem aging [4]. Ntawm qhov tod tes, exogenous aging yog kho los ntawm lwm yam (xws li, overexposure rau ultraviolet hluav taws xob (photoaging), lub ntiajteb txawj nqus, haus luam yeeb, muaj kuab paug, thiab noj zaub mov tsis zoo)[5]. Nyob rau hauv ob qho tib si txheej txheem kev laus, tab sis tshwj xeeb tshaj yog exogenous ib qho, nws tau txais zoo tias qhov nce oxidative kev nyuaj siab, tshwm sim los ntawm reactive oxygen hom (ROS) thiab tshwm sim los ntawm unbalance ntawm ROS ntau lawm thiab antioxidant tiv thaiv, plays lub luag hauj lwm tseem ceeb [{{3} }]. Nrog lub hnub nyoog, kuj tseem muaj qhov txo qis hauv lub peev xwm ntawm tib neeg cov tawv nqaij los kho DNA puas, uas ua rau cov tawv nqaij laus ib yam nkaus [8].Cistanche Extract Anti RadiationYog li, cov txheej txheem kev laus ua rau muaj ntau yam kev hloov pauv hauv cov tawv nqaij, nrog rau kev cuam tshuam ntawm nws cov qauv thiab kev ua haujlwm [9], ua rau pom cov tsos mob xws li wrinkling, dryness, poob ntawm elasticity, thinning, ntxhib ntxhib ntxhib los mos, los yog pigmentation tsis xwm yeem [7,10] . Yog li ntawd, nrog rau kev loj hlob mus rau kev noj qab haus huv thiab kev noj qab haus huv, nyob rau hauv uas cov tawv nqaij noj qab haus huv thiab zoo nkauj kuj muaj, kev loj hlob ntawm cov tshuaj pleev ib ce incorporating bioactive compounds los tiv thaiv los yog attenuate ntawm daim tawv nqaij laus thiab nws cov tsos mob sab nraud, yog li txhim kho daim tawv nqaij tsos, tau nyob rau hauv lub forefront ntawm kev tshawb fawb thiab innovation nyob rau hauv kev lag luam tshuaj pleev ib ce nyob rau hauv lub xyoo dhau los. Hauv cov ntsiab lus no, thiab ntawm cov tshuaj pleev ib ce rau kev kho mob, lub ntsej muag zoo nkauj, thiab tshwj xeeb tshaj yog daim npog ntsej muag, yog kev lag luam loj hlob, nrog rau qhov kev cia siab ntawm kev loj hlob txhua xyoo (CAGR) ntawm 8.1 feem pua ​​​​ntawm 2019 txog 2027 [11]. Hom npog ntsej muag no tshwj xeeb tshaj yog rau cov neeg siv khoom niaj hnub no, feem ntau vim nws daim ntawv thov yooj yim thiab yooj yim, siv ceev, thiab ua tau zoo]12. Ntau cov ntaub ntawv tuaj yeem siv los ua kev txhawb nqa ntawm cov ntawv npog no, xws li cov khoom siv hluavtaws (xws li, poly (vinyl cawv) thiab silicone, ntaub (xws li, tsis-woven thiab paj rwb), hydrogels (xws li, collagen- thiab silk-based hydrogels). ) lossis cellulose nanofibers, xws li cov kab mob nanocellulose (BNC) [13]. Nrog rau qhov kev thov ntau ntxiv rau cov khoom tu tawv nqaij, BNC tau txais qhov tseem ceeb thiab tam sim no yog cov khoom siv zoo tshaj plaws bio-based, twb tau tshawb nrhiav, thiab kev lag luam los ntawm qee qhov ntawm cov tuam txhab tshuaj pleev ib ce saum toj kawg nkaus (piv txwv li, Lancome, Elizabeth Arden, thiab DHC) [14] uas ua raws li cov kev xav tau ntawm cov neeg siv khoom ntsuab.

Bacterial nanocellulose is an extracellular polysaccharide produced by several non-pathogenic bacteria from different genera (e.g., Komagataeibacter (formerly Gluconacetobacter), Agrobacterium, Rhizobium, Escherichia, and Aerobacter), through fermentation in the presence of oxygen and carbon sources(e.g., glucose)[1516]. BNC is synthesized in the air-culture medium interface as an ultrafine 3D nanofiber network, showing high water content(>90 feem pua), siab hydrophilicity, thiab ib tug nanoporous qauv [17]. Thaum tsim nyob rau hauv cov kab lis kev cai zoo li qub, BNC tau txais raws li gelatinous hydrogel zoo li daim nyias nyias nrog cov tuab sib txawv thiab cov duab ntawm kab lis kev cai (viz. hauv situ moldability), uas yog qhov zoo dua thaum xav tau cov duab ua ntej [18]. Cov txheej txheem tshwj xeeb ntawm BNC kuj txhawb siab crystallinity thiab purity, zoo kawg li txhua yam muaj zog, thiab thermal stability [19]. Dhau li ntawm biodegradable, BNC kuj tau ua kom pom cov tawv nqaij zoo thiab biocompatibility hauv ntau qhov kev tshawb fawb vivo [20-22], ua rau nws yog ib qho khoom siv zoo tshaj plaws rau cov khoom tu tawv nqaij. Nyob rau hauv no hais txog, thiab vim hais tias ntawm nws cov ntxeem tau nanostructure, kev siv thiab kev ua tau zoo ntawm BNC raws li ib tug neeg nqa khoom rau kev xa ntawm daim tawv nqaij active compounds tau lub hom phiaj ntawm ob peb ua hauj lwm, piv txwv li, rau tus me nyuam ntawm caffeine [23], silk-sericin [24], retinol [25], lossis rutin [26]. Rau kev nyeem ntawv ntxiv, kev tshuaj xyuas tsis ntev los no muab cov ntsiab lus dav dav ntawm kev siv ntau yam thiab siv tau ntawm BNC hauv cov tshuaj pleev ib ce ntsuab, suav nrog ua cov khoom siv ntawm daim tawv nqaij [27].

cistanche tuaj yeem tiv thaiv kev laus

Ntuj bioactive compounds yog dav siv hauv tshuaj pleev ib ce tiv thaiv kev laus [28].cistanche tshuaj ntsuabCov nroj tsuag rho tawm, tshwj xeeb, yog cov nplua nuj ntawm cov tshuaj bioactive (piv txwv li, polyphenols, terpenoids, vitamins, thiab lwm yam) uas muaj peev xwm ua tau ntau yam ntawm daim tawv nqaij, nrog rau cov tshuaj tua kab mob antioxidant thiab tshuaj tua kab mob ntawm tyrosinase inhibition los yog cov txiaj ntsig tseem ceeb tshaj tawm txog tam sim no [ 29] ib. Yog li ntawd, muab cov qauv tam sim no rau cov khoom siv ntuj tsim hauv cov tshuaj pleev ib ce, cov nroj tsuag extracts tshwm sim ntawm cov khoom xyaw zoo tshaj plaws rau kev tsim cov khoom tu tawv nqaij. Tsis tas li ntawd, kev koom ua ke ntawm natural extracts rau hauv BNC daim nyias nyias kuj tau tshawb xyuas rau lub hom phiaj kom zoo nkauj. Piv txwv yog cov haujlwm tshaj tawm txog kev koom ua ke ntawm propolis extract, paub tias muaj tshuaj tua kab mob thiab kev ua haujlwm astringent[13,30], rho tawm los ntawm oat thiab rosemary nrog moisturizing los [13], los yog cov tshuaj pleev ib ce tsim los ntawm Adansonia digitata txiv hmab txiv ntoo, Hibiscus sabdariffa paj, Coffea arabica seedcake, Kigelia Africana txiv hmab txiv ntoo, thiab Acacia thiab Crocus chrysanthus noob rho tawm, nrog anti-aging zog, namely wrinkle-smoothing [21]. Tsis ntev los no, kev tshuaj xyuas tau ua qhov kev pom tseem ceeb rau qhov tseem ceeb ntawm kev koom nrog BNC nrog cov nroj tsuag phenolics los tiv thaiv UV-induced tawv nqaij puas tsuaj [31].

Eucalyptus globulus Labill. tsob ntoo evergreen yog qhov paub zoo ntawm cov tshuaj bioactive, uas yog cov tshuaj phenolic xws li phenolic acids, flavonoids, lossis hydrolyzable tannins [32-36]. Cov tshuaj tua kab mob antioxidant thiab tshuaj tua kab mob ntawm E.globulus extracts uas muaj cov tshuaj no tau pom nyob hauv ntau qhov kev tshawb fawb [34,35]. Hais txog daim ntawv thov nyob rau hauv cov tshuaj pleev ib ce, ib txoj kev tshawb no tsis ntev los no qhia tau hais tias cov nyhuv tiv thaiv, ob qho tib si nyob rau hauv vitro (nyob rau hauv tib neeg dermal fibroblasts) thiab nyob rau hauv vivo (hairless nas), ntawm ib tug 50 feem pua ​​​​ethanolic extract los ntawm qhuav coj mus muag E.globulus biomass tiv thaiv UV-induced photoaging. , nrog cov txiaj ntsig tau zoo hais txog kev tiv thaiv ntawm wrinkle tsim thiab tawv nqaij qhuav [37]. Txawm li cas los xij, rau qhov zoo tshaj plaws ntawm peb txoj kev paub, E.globulus extracts yeej tsis tau tshawb pom, ua ke nrog polymeric substrate, los tsim bioactive daim tawv nqaij qhov ncauj qhov ntswg rau kev tiv thaiv kev laus ntawm daim tawv nqaij.

Nrog rau qhov kev xav no, lub hom phiaj ntawm txoj haujlwm tam sim no yog los tsim BNC mem-branes loaded nrog ib qho aqueous extract tau los ntawm hydro-distillation (hydro-distillation extract, HDE) ntawm E.globulus nplooj. Hydro-distillation yog ib txoj kev uas feem ntau siv los ntawm kev lag luam rau kev rho tawm cov roj yam tseem ceeb [38]. Tsis tas li ntawd, glycerol (G), uas yog siv dav siv hauv cov tshuaj pleev ib ce [39], kuj tau muab tso rau hauv BNC daim nyias nyias (7.5 mg cm-2) ​​kom lawv yooj yim dua thiab, tib lub sij hawm, lawv conformability daim tawv nqaij. Yog li, lub hom phiaj ntawm txoj haujlwm no yog kom tau txais cov khoom siv bio-raws li tag nrho nrog cov khoom siv ua haujlwm rau daim ntawv thov muaj peev xwm ua daim npog ntsej muag tiv thaiv kev laus. BNC daim nyias nyias ntim nrog ntau npaum li cas ntawm cov hydro-distillation extract tau pom nyob rau hauv cov nqe lus ntawm lawv morphology, txhua yam khoom, thermal stability, thiab noo noo-uptake peev xwm. Tsis tas li ntawd, cov tshuaj tua kab mob antioxidant ntawm cov kab mob sib txawv kuj tau soj ntsuam, nrog rau lawv cov cytotoxicity hauv vitro (hauv tib neeg keratinocytes thiab nas fibroblasts cell kab). Tsis tas li ntawd, cov tshuaj tiv thaiv antioxidant stability tom qab 3 lub hlis ntawm kev cia, hauv qhov tsaus ntuj, ntawm 22-25 degree thiab 52 feem pua ​​​​ntawm cov av noo (RH) lossis ntawm 40 degree thiab 75 feem pua ​​RH ntawm daim nyias nyias nrog 2 ug cm{{19} } ntawm HDE raug soj ntsuam. Thaum kawg, hauv vitro tiv thaiv kev ua haujlwm ntawm BNC daim nyias nyias nrog 2 ug cm -2 ntawm HDE tau tshawb xyuas ntxiv.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Tshuaj thiab khoom siv

Citric acid (≥99.5%), dimethyl sulfoxide (DMSO) (≥99.0%), disodium hydrogen phosphate(≥99.0%), glucose(≥99.5%), glycerol(≥99.5%), magnesium nitrate hexahydrate (≥99.0%), potassium chloride(≥99.0%), potassium dihydrogen phosphate (≥99.0%), potassium sulphate (>99.0%), sodium bicarbonate (>99.5%), sodium chloride (>99. tshaj los yog sib npaug rau 97.5 feem pua) tau yuav los ntawm Sigma-Aldrich (Lisbon, Portugal). Tag nrho lwm cov tshuaj yog nyob rau qib kuaj.cistanche noov loj hlobCalcium chloride, Dulbecco's Modified Eagle's Medium (DMEM), etoposide, Folin-Ciocalteu's phenol reagent, magnesium chloride, sodium pyruvate, trypsin-EDTA kua, N-(2-}hydroxyethyl)piperazine-N'-({{6}) }ethane sulfonic acid), 4-(2-hydroxyethyl piperazine-1-ethane sulfonic acid (HEPES), thiab 2,2-diphenyl-1-picrylhydrazyl(DPPH) yog muab los ntawm Sigma-Aldrich (Lisbon, Portugal). Peptone thiab poov xab extract tau txais los ntawm Himedia Laboratories GmbH (Einhausen, Lub teb chaws Yelemees).

Cov qauv los ntawm BNC daim ntawv lag luam daim npog ntsej muag tau siv rau hauv kev tshuaj xyuas cov khoom siv thiab cov dej noo, rau kev sib piv. BNC lag luam daim ntawv npog ntsej muag (Superstart Probiotic Boost Skin Renewal Biocellulose Mask, Elizabeth Arden, New York, NY, USA) tau piav qhia tias yog muab los ntawm cov txiv maj phaub dej ntuj thiab muab tso rau hauv cov tshuaj pleev ib ce nrog cov khoom siv rov ua dua tshiab ntawm daim tawv nqaij, nrog rau lwm cov khoom xyaw, probiotics (Lactococcus ferment lysate, Lactobacillus), cov nroj tsuag rho tawm (Althea rose paj extract, Polymnia sonchifolia cag kua txiv, Crithmum maritimum extract), caffein, thiab humectants, xws li glycerol, hyaluronic acid, los yog polyethylene glycol (PEG){0} . Cov qauv ntawm BNC-kev lag luam ntawv tau qhuav hauv qhov cub qhov cua ntawm 358C ua ntej siv.cistanche salsa cov txiaj ntsigFresh E.globulus Labill. nplooj tau sau los ntawm cov ntoo laus los ntawm kev cog qoob loo ntawm Lub Tuam Txhab Navigator ze Aveiro ntawm Sever do Vouga (Bracal)(Aveiro, Portugal) thiab xa mus rau cov txheej txheem hydro-distillation kom txog thaum tiav cov roj tseem ceeb feem (2-3 h) siv cov cuab yeej hloov kho Clevenger. Thaum kawg, aqueous extract, HDE, tau zoo tu qab thiab lyophilized, khaws cia, thiab tiv thaiv los ntawm lub teeb nyob rau hauv ib tug desiccator, nyob rau hauv chav tsev kub, mus txog rau thaum siv [40].

2.2. Tag nrho Phenolic Cov ntsiab lus ntawm HDE

Tag nrho cov ntsiab lus phenolic (TPC) ntawm HDE tau txiav txim siab siv Folin-Ciocalteu txoj kev, raws li tau piav qhia nyob rau lwm qhov [41], nrog qee qhov kev hloov kho. Luv luv, nyob rau hauv ib tug 96-zoo phaj, 15{19}} μL ntawm Folin-Ciocalteu reagent yav tas los diluted (1∶10, o / ø) nrog dej thiab 120 L ntawm aqueous sodium carbonate tov (75 g L- ) tau ntxiv rau 30 μL ntawm cov tshuaj aqueous HDE hauv cov ntsiab lus xws li 1.95 ugs txog 500 ug mL -1 ntawm cov extract. Cov tshuaj tiv thaiv sib xyaw yog incubated nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub rau 60 min. Tom qab lub sij hawm incubation, lub absorbance yog ntsuas ntawm 760 nm tiv thaiv ib tug dawb paug (dej es tsis txhob extract) nyob rau hauv ib tug Thermo Scientific MultiskanTM FC microplate nyeem ntawv (Thermo Fisher Scientific Inc., Waltham, MA, USA). TPC raug xam raws li gallic acid sib npaug (GAE) los ntawm calibration nkhaus ntawm gallic acid txheem daws teeb meem (5.13-205.0 ug mL-1) thiab qhia raws li mg ntawm GAE ntawm lyophilized extract. Qhov kev ntsuam xyuas tau ua tsib zaug, thiab txhua tus qauv tau txheeb xyuas hauv triplicate.

2.3. Kev tsim khoom ntawm BNC Membranes

BNC daim nyias nyias tau tsim nyob rau hauv peb lub chaw soj nstuam siv Hestrin-Schramm (HS) kua kab lis kev cai nruab nrab (20g L-1glucose, 5g L-1 peptone, 5g Ll poov xab extract, 2.7g L -1 disodium hydrogen phosphate, 1.15 g L-Citric acid, pH5) inoculated nrog cov kab mob acetic acid Gluconacetobacter sacchari raws li kab lis kev cai zoo li qub [42]. Tom qab 4-6 hnub kab lis kev cai ntawm 30 degree, BNC daim nyias nyias tau sau thiab kho ob zaug nrog 0.5 M NaOH ntawm 80 degree rau 30 min. Tom qab ntawd, daim nyias nyias tau ntxuav ob peb zaug nrog dej distilled kom tshem tawm cov seem nruab nrab thiab cov kab mob hlwb. Thaum kawg, BNC daim nyias nyias tau ntxuav nrog 1 feem pua ​​​​sodium hypochlorite aqueous tov tom qab rov qab ntxuav nrog dej distilled kom txog thaum nruab nrab pH. Purified membranes tau muab cia rau hauv dej ultrapure ntawm ntxiv 4 degree kom txog thaum siv.

2.4. Kev npaj ntawm BNC-G-HDE Membranes

BNCmembranes ntub dej (diameter∶ ca.7.0±0.5 cm; thickness∶ 7000 ± 1000 μm) tau ntim nrog cov tshuaj sib txawv ntawm HDE (qhia raws li qhov loj ntawm HDE ib thaj tsam ntawm daim nyias nyias) thiab glycerol (7.5 mg cm-2) ​​(Table 1), siv txoj kev impregnation. Succinctly, ntub BNC daim nyias nyias tau hnyav (kwv yees li 180 mg ntawm BNC qhuav) thiab ntws los ntawm tes-nias nrog cov ntawv kuaj-qib nqus nqus kom txog thaum cov dej txo qis rau ze li 40 feem pua ​​(kwv yees los ntawm kev poob phaus). Tom qab ntawd, daim nyias nyias tau muab tso rau hauv 5 mL ntawm cov tshuaj aqueous uas muaj cov koob tshuaj ntawm ob qho tib si HDE thiab glycerol thiab khaws cia nyob rau hauv chav tsev kub kom txog thaum ua tiav cov tshuaj HDE-G. Thaum kawg, BNC daim nyias nyias tau qhuav (hauv Petri tais diav) ntawm 35 degree hauv qhov cub qhov cua (Venticell Eco kab, MMM pawg, Planegg, Lub teb chaws Yelemees) tsawg kawg 20 teev. Qhuav daim nyias nyias tau khaws cia thiab tiv thaiv los ntawm lub teeb nyob rau hauv ib tug desiccator ntawm chav tsev kub kom txog rau thaum siv. Rau kev sib piv, cov ntshiab BNC daim nyias nyias (tsis muaj cov extract thiab glycerol) tau qhuav raws li tau piav qhia dhau los, thiab cov khoom ntim nrog glycerol (BNC-G) tau npaj ua raws li cov txheej txheem tib yam.

2.5. Characterization ntawm Membranes

2.5.1.Thickness

Lub thickness ntawm cov ntaub so ntswg qhuav tau ntsuas nyob rau hauv tsib qhov chaw random siv lub tes tuav digital micrometer (Mitutoyo Corporation, Tokyo, Nyiv) nrog qhov tseeb ntawm 1 μm

2.5.2. Morphology

Qhov saum npoo thiab ntu ntu (cryo-fractured) morphologies ntawm daim nyias nyias tau txheeb xyuas los ntawm kev tshuaj xyuas electron microscopy (SEM). Micrographs tau txais los ntawm kev siv lub tshuab ntsuas hluav taws xob siab (HR-FESEMSU 70 Hitachi, Tokyo, Nyiv) ua haujlwm ntawm 4.0 kV. Ua ntej yuav cov duab, cov qauv tau muab tso rau ntawm kev txhawb nqa steel thiab coated nrog carbon.

2.5.3. Mechanical Performance

Kev ua tau zoo ntawm cov membranes hauv qhuav thiab ntub dej (80 feem pua ​​​​ntawm ya raws) tau soj ntsuam los ntawm kev ntsuas tensile. Cov dej noo ntawm cov ntaub ntub dej tau txhais raws li cov ntsiab lus noo noo tau txiav txim siab hauv kev lag luam BNC. Tensile xeem tau ua nyob rau hauv uniaxial Instron 5564 kuaj tshuab (Instron Corporation, Norwood, MA, USA) nyob rau hauv lub traction hom ntawm ib tug crosshead tshaj tawm ntawm 10 mm min-I siv 500 N static load cell. Tag nrho cov kev ntsuas tau ua nyob rau hauv tsawg kawg yog tsib replicates siv cov duab plaub (5 × 1 cm²) thiab ib tug gauge ntev ntawm 30 mm. Kev ntxhov siab （MPa） thiab strain （feem pua） curves tau npaj, thiab Young's modulus, tensile zog, thiab elongation ntawm kev tawg tau txiav txim siab siv Instron BlueHill 3 software. Hauv cov kev ntsuam xyuas no, cov qauv ntawm kev lag luam BNC lub ntsej muag lub ntsej muag kuj tau sim hauv tib yam kev mob, rau kev sib piv.

2.5.4 ib. Thermal stability

Thermogravimetric tsom xam (TGA) tau ua nrog SETSYS Setaram TGA analyzer (SITARAM Instrumentation, Lyon, Fabkis) nruab nrog lub platinum cell. Cov qauv tau ua kom sov los ntawm chav tsev kub txog 800C ntawm qhov tsis tu ncua ntawm 10 degree min-1 nyob rau hauv ib qho chaw nitrogen.

2.5.5. Moisture Uptake Capacity

Lub peev xwm nqus dej noo ntawm txhua daim nyias nyias tau soj ntsuam los ntawm kev tso cov qauv qhuav (2 × 2 cm-) ntawm txhua daim nyias nyias nyob rau hauv lub desiccator, ntawm chav tsev kub, nrog cov av noo ntawm ca. 52 feem pua ​​​​yog siv cov saturated magnesium nitrate aqueous tov (52.89 ± 0.22 feem pua ​​​​ntawm 25 degree) [43]. Cov qauv coj los ntawm lub tshuab desiccator thiab hnyav tom qab 0.5 h, 1 h, 2.5 h, 24 h, thiab 48 h. Txhua daim nyias nyias tau kuaj hauv triplicate. Moisture uptake tau xam raws li hauv qab no:

qhov twg Whois tus qauv qhov hnyav thawj zaug, thiab Wwis qhov hnyav ntawm txhua lub sijhawm.

2.6. Hauv Chermico Antioxidant Activity

Kev ua haujlwm antioxidant ntawm HDE-loaded membranes tau kwv yees siv DPPH dawb radical scavenging txoj kev, ua raws li cov txheej txheem tau tshaj tawm yav dhau los [44], nrog qee qhov kev hloov kho. Cov tshuaj tiv thaiv yog raws li qhov txo qis ntawm qhov nqus ntawm DPPH cov tshuaj uas tshwm sim los ntawm cov radical scavenging los ntawm cov tshuaj tiv thaiv antioxidant, nrog rau qhov kev hloov xim ntawm cov tshuaj DPPH los ntawm ntshav mus rau daj ntseg [45]. Luv luv, cov qauv (2 × 5 cm²) ntawm txhua daim nyias nyias tau ntxiv rau 3.75 mL ntawm ethanol (EtOH) thiab phosphate-buffered saline (PBS) daws (60∶40; pH 5.5). Tom qab ntawd, 250 μL ntawm DPPH (1 mM) kev daws teeb meem hauv ethanol tau ntxiv, thiab cov txiaj ntsig sib xyaw tau muab tso rau hauv qhov tsaus ntuj nrog kev sib xyaw maj mam (100rpm) ntawm 22 degree rau 0.5 h, 1 h, thiab 2.5h, thiab tom qab ntawd qhov nqus ntawm 517nm tau nyeem tawm tsam qhov khoob (EtOH: PBS1 × pH5.5) siv MultiskanTM FC microplate nyeem ntawv (Thermo Scientific, Waltham, Massachuses, EUA). Rau kev sib piv, cov tshuaj tiv thaiv sib xyaw uas muaj HDE diluted hauv EtOH: PBS1 × pH5.5 ntawm qhov kawg concentration sib npaug rau qhov siab tshaj plaws ntawm HDE uas tuaj yeem tawm ntawm txhua daim nyias nyias (HDE 1: 2.5 ug mL-1; HDE1. 5∶3.75 ug mL-1; HDE 2∶5.0 ug mL-1; HDE 3∶7.5 ug mL-) kuj tau suav nrog hauv qhov kev xeem. Kev tswj muaj EtOH: PBS 1 × pH5.5 Nrog DPPH thiab tsis muaj daim nyias nyias, peb qhov kev soj ntsuam ywj pheej tau ua rau txhua tus qauv, DPPH radical scavenging kev ua tau suav los ntawm kev nqus ntawm txhua tus qauv (Ib qho qauv) nrog rau kev tswj DPPH absorbance (ApppH) raws li hauv qab no:

2.7. Kev ntsuas kev ruaj ntseg ntawm BNC-G-HDE2 Membrane nyob rau hauv Cia

Raws li "Cov Lus Qhia ntawm Kev Ntsuam Xyuas Kev Ruaj Ntseg rau cov khoom lag luam kom zoo nkauj" [46], kev ruaj ntseg cia ntawm BNC-G-HDE2 tau soj ntsuam. Rau qhov no, cov qauv (14 cm²) ntawm BNC-G (raws li kev tswj) thiab BNC-G-HDE2 daim nyias nyias tau muab tso rau hauv iav vials thiab khaws cia rau 3 lub hlis hauv qhov tsaus ntuj ntawm qhov kev cia siab ib txwm muaj, ntawm chav tsev kub ({{10). }} degree ) thiab 52 feem pua ​​​​RH (ib yam mob kuv, siv cov tshuaj magnesium nitrate saturated [43]) thiab ntawm qhov ceev ceev ntawm 40 degree C thiab 75 feem pua ​​RH (ib yam mob kuv, siv sodium chloride saturated tov [43] ). Cov av noo txheeb ze tau saib xyuas ib ntus uas siv lub thermohydrometer kom ntseeg tau tias cov av noo tas li thaum lub sijhawm khaws cia tag nrho. Tag nrho cov qauv tau txheeb xyuas ua ntej thiab tom qab 1,2, thiab 3 lub hlis ntawm kev khaws cia txog lawv cov tshuaj tiv thaiv antioxidant (raws li tau piav qhia hauv Tshooj 2.6). Lub macroscopic nam ntawm daim nyias nyias kuj tau sau tseg. Rau txhua tus mob, tag nrho cov qauv tau txheeb xyuas hauv triplicate.

2.8. Hauv Vitro Biological Assay

2.8.1 ib. Cell Culture

Tib neeg keratinocytes (HaCaT, los ntawm CLS, Cell Lines Service, Eppelheim, Lub teb chaws Yelemees) thiab nas fibroblasts (NIH/3T3, ATCC CRL-1658, Manassas, VA, USA) cell kab tau coj los siv Dulbecco's Modified Eagle's Medium (DMEM) , ntawm 37 degree , nyob rau hauv ib tug humidified5 feem pua ​​CO2-95 feem pua ​​ntawm huab cua. Qhov nruab nrab tau ntxiv nrog 10 feem pua ​​​​(o / v) thaum tshav kub kub-inactivated fetal bovine serum (FBS), 1 feem pua ​​(o/ø) cwj mem/strep, 3.7 g L-Sodium bicarbonate, thiab 1 mM sodium pyruvate. Cells raug tshem tawm nrog trypsin-EDTA daws 1 ×.

2.8.2 ib. Cytotoxicity Assay

Lub cytotoxicity ntawm daim nyias nyias tau soj ntsuam los ntawm kev ntsuas MTT txo. Cov qauv (2.5 cm²) ntawm daim nyias nyias (BNC-G, BNC-G-HDE1, BNC-G-HDE1.5, BNC-G-HDE2, BNC-G-HDE3) tau sterilized ob zaug los ntawm UV hluav taws xob ntawm txhua sab thaum 20 min thiab ntxiv incubated, rau 24 h, nyob rau hauv 2.5 mL ntawm tiav DMEM nruab nrab ntawm 37 degree nyob rau hauv ib tug humidified 5 feem pua ​​CO, -95 feem pua ​​​​ntawm huab cua, los npaj txhua daim nyias nyias.

Lub caij no, 2 × 10 thiab 1 × 104 lub hlwb / lub qhov dej tau raug cog rau hauv 96- cov phaj zoo rau HaCaT thiab NIH / 3T3 hlwb, raws li. Tom qab 24 teev, tib lub ntim ntawm daim nyias nyias tau siv los hloov cov kab lis kev cai nruab nrab, thiab cov hlwb tau ntxiv rau 24 teev ntawm 37 degree. Raws li kev tswj hwm, cov hlwb raug kho tib yam li tau piav qhia rau cov qauv tab sis tsuas yog nthuav tawm rau DMEM nruab nrab. Tom qab 24 teev, qhov nruab nrab raug tshem tawm, thiab cytotoxicity tau txiav txim siab raws li tau piav qhia yav dhau los [47]. Luv luv, cov tshuaj tshiab ntawm MTT (0.5 mg L-1) tau npaj rau hauv Krebs nruab nrab (pH 7.4) tau ntxiv thiab incubated ntawm 37 degree rau 2h (HaCaT hlwb) lossis 4h (NIH/3T3 hlwb). Tom qab ntawd, cov tshuaj MTT tau hloov los ntawm DMSO thiab incubated rau l0 min, nrog co co, kom yaj tag nrho cov formazan muaju. Tom qab incubation, qhov nqus tau ntsuas ntawm 570 nm hauv spectrophotometer (SLT spectra I). Cov txiaj ntsig ntawm 3 qhov kev sim ywj pheej, nrog 3 replicates txhua, tau qhia raws li qhov feem pua ​​(feem pua) ntawm tus nqi absorbance tau txais los ntawm kev tswj thiab graphically nthuav tawm raws li feem pua ​​​​ntawm MTT txo.

2.8.3 ib. Anti-Senescence Activity

Kev ua haujlwm los tiv thaiv kev laus ntawm BNC-G-HDE2 daim nyias nyias tau raug tshuaj xyuas siv -galactosidase (-gal) staining assay. BNC-G membrane tau siv los ua kev tswj hwm. Txhawm rau npaj cov ntaub so ntswg tshem tawm, cov qauv ua kom tsis muaj menyuam (10 cm-) tau muab tso rau hauv 10 mL ntawm ua tiav DMEM nruab nrab raws li tau piav qhia yav dhau los rau kev ntsuas cytotoxicity.

NIH/3T3 hlwb tau muab noob rau hauv 12- cov phiaj zoo ntawm qhov ceev ntawm 2.5 × 104 hlwb / qhov zoo thiab tso cai rau stabilize rau 24 teev. Tom qab ntawd, 12.5 μM etoposide tau siv los ua kom cov cellular senescence hauv NIH / 3T3 rau 24 teev. Etoposide-stimulated hlwb raug kho nrog BNC-G-HDE2 membrane extract rau lwm 24 teev.cistanche tubulosa noj redditRaws li kev tswj hwm, cov hlwb tsis kho tau raug tsim nrog DMEM nruab nrab lossis nrog cov khoom rho tawm ntawm BNC-G thiab BNC-G-HDE daim nyias nyias. Tom qab incubation, kab lis kev cai nruab nrab raug tshem tawm, thiab hlwb raug ntxuav nrog PBS thiab cim nrog -gal tov npaj raws li piav los ntawm cov chaw tsim tshuaj paus (Cell Signaling Technology, Danvers, MA, USA). Kev tsom xam ntawm cov hlwb zoo rau kev laus tau ua tiav ntawm 20 × magnification siv lub tshuab ntsuas dav dav (Carl Zeiss, Oberkochen, Lub teb chaws Yelemees). Tsawg kawg yog 3 qhov kev sim ywj pheej tau ua nyob rau hauv replicate, thiab feem pua ​​​​ntawm -gal-zoo hlwb tau txiav txim siab siv plaub daim duab microscopic.

2.9. Kev txheeb cais

Hauv kev kho mob ntawm cov txiaj ntsig los ntawm tus cwj pwm, kev ua haujlwm antioxidant, thiab kev ntsuas kev ruaj ntseg ntawm daim nyias nyias, ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) ua raws li Tukey qhov kev sim tau siv los ntsuas qhov tseem ceeb. Cov txiaj ntsig tau qhia raws li qhov nruab nrab ± tus qauv sib txawv ntawm qhov nruab nrab. Hais txog kev soj ntsuam hauv vitro, qhov normality ntawm cov ntaub ntawv faib tau raug soj ntsuam los ntawm D'Agostino-Pearson thiab Shapiro-Wilk normality tests. Kev sib piv ntawm cov pab pawg tau ua los ntawm ANOVA ua raws li Dunnett qhov kev xeem tom qab hoc lossis tsis tau ua khub Cov Tub Ntxhais Kawm 't-test. Cov txiaj ntsig tau nthuav tawm raws li qhov nruab nrab ± tus qauv kev ua yuam kev ntawm qhov nruab nrab ntawm tus lej qhia kev sim.

Qhov tseem ceeb tau txais ntawm qhov tseem ceeb ntawm p<0.05. all="" statistical="" calculations="" were="" performed="" using="" graphpad="" prism="" software(8.0.2,="" graphpad="" software="" inc.,="" san="" diego,="" ca,="">

Kab lus no yog muab rho tawm los ntawm Cov Ntaub Ntawv 2022, 15, 1982. https://doi.org/10.3390/ma15051982 https://www.mdpi.com/journal/materials