Cosmeceutical Potential Of Extracts Derived From Fishery Industry Residues: Sardine Wastes And Codfish Frames Part 1
Jun 29, 2023
Abstract: Kev lag luam nuv ntses tsim cov khoom pov tseg ntau (20-75 feem pua (w/w) ntawm tag nrho cov ntses ntses hnyav). Kev rov qab los ntawm bioactive compounds los ntawm residues thiab lawv kev koom ua ke hauv cov tshuaj pleev ib ce sawv cev rau kev lag luam pheej hmoo thiab tuaj yeem ua rau muaj txiaj ntsig zoo ntawm kev lag luam. Hauv kev ua haujlwm no, cov khoom noj muaj protein ntau tau txais los ntawm cov thev naus laus zis siab (supercritical CO2 thiab dej subcritical) los ntawm sardine (Sardina pilchardus) pov tseg thiab codfish (Gadus morhua) thav duab tau qhia txog lawv lub peev xwm cosmeceutical. Antioxidant, anti-inflammatory thiab antibacterial kev ua ub no raug soj ntsuam los ntawm tshuaj lom neeg (ORAC assay), enzymatic (inhibition ntawm elastase thiab tyrosinase), antimicrobial susceptibility (Klebsiella pneumoniae, Staphylococcus aureus, thiab Cutibacterium acnes) thiab cell-based (hauv- Caatinocytes). . Sardine extracts nthuav tawm cov tshuaj tua kab mob siab tshaj plaws, thiab ntu tau txais los ntawm kev siv cov extraction kub siab dua (250 ◦C) thiab tsis muaj cov kauj ruam defatting tau pom qhov qis tshaj qhov tsawg kawg nkaus inhibitory concentration (MIC) qhov tseem ceeb (1.17; 4.6; {{24}). }.59 mg/mL rau K. pneumoniae, S. aureus, thiab C. acnes, ntsig txog). Codfish cov qauv muab rho tawm ntawm qhov kub thiab txias (90 ◦C) yog cov tshuaj tiv thaiv kab mob zoo tshaj plaws (qhov siab ntawm 0.75 mg / mL txo IL-8 thiab IL{20}} qib los ntawm 58 feem pua thiab 47 feem pua, raws li , txheeb ze rau qhov zoo tswj). Threonine, valine, leucine, arginine, thiab tag nrho cov ntsiab lus ntawm cov protein nyob rau hauv cov extracts tau qhia kom muaj kev sib raug zoo nrog cov kev qhia txog bioactivities (R2 Ntau dua lossis sib npaug rau 0.7). Cov txiaj ntsig no txhawb nqa cov peev txheej ntawm kev siv cov extracts los ntawm kev lag luam nuv ntses pov tseg hauv cov khoom siv tshuaj pleev ib ce nrog cov dej num bioactive.
Glycoside ntawm cistanche tuaj yeem ua rau muaj kev ua haujlwm ntawm SOD hauv plawv thiab daim siab cov ntaub so ntswg, thiab txo cov ntsiab lus ntawm lipofuscin thiab MDA hauv txhua cov ntaub so ntswg, tshem tawm ntau yam reactive oxygen radicals (OH-, H₂O₂, thiab lwm yam) thiab tiv thaiv DNA puas. los ntawm OH-radicals. Cistanche phenylethanoid glycosides muaj peev xwm tshem tawm cov dawb radicals, muaj peev xwm txo tau ntau dua li cov vitamin C, txhim kho cov haujlwm ntawm SOD hauv cov phev ncua, txo cov ntsiab lus ntawm MDA, thiab muaj qee yam kev tiv thaiv ntawm cov phev ua haujlwm. Cistanche polysaccharides tuaj yeem txhim kho kev ua haujlwm ntawm SOD thiab GSH-Px hauv erythrocytes thiab ntsws cov ntaub so ntswg ntawm cov nas senescent sim los ntawm D-galactose, nrog rau txo cov ntsiab lus ntawm MDA thiab collagen hauv lub ntsws thiab ntshav, thiab nce cov ntsiab lus ntawm elastin, muaj Cov nyhuv scavenging zoo ntawm DPPH, ncua lub sijhawm ntawm hypoxia hauv cov nas senescent, txhim kho cov haujlwm ntawm SOD hauv cov ntshav, thiab ncua lub physiological degeneration ntawm lub ntsws hauv kev sim cov nas nrog cellular morphological degeneration, kev sim tau pom tias Cistanche muaj peev xwm antioxidant zoo. thiab muaj peev xwm los ua ib qho tshuaj tiv thaiv thiab kho cov kab mob ntawm daim tawv nqaij laus. Nyob rau tib lub sijhawm, echinacoside hauv Cistanche muaj lub peev xwm tseem ceeb txhawm rau txhawm rau DPPH dawb radicals thiab muaj peev xwm tshem tawm cov pa oxygen reactive thiab tiv thaiv dawb radical-induced collagen degradation, thiab kuj muaj kev kho zoo ntawm thymine dawb radical anion puas.
Nyem rau ntawm Cistanche Tubulosa Ntxiv
【Yog xav paub ntxiv:george.deng@wecistanche.com / WhatApp:86 13632399501 】
Ntsiab lus: ntses pov tseg kwj deg valorisation; kev ua haujlwm antioxidant; anti-inflammatory kev ua; antimicrobial kev ua haujlwm; anti-aging; anti-hyperpigmentation; cosmeceuticals
1. Taw qhia
Nrog rau kev tshawb nrhiav tas li rau kev tsim kho tshiab, tshwj xeeb tshaj yog rau cov khoom xyaw nquag, kev lag luam tshuaj pleev ib ce tau loj hlob thiab tau pom lub hom phiaj los hloov cov roj av-derived Cheebtsam mus rau pem hauv ntej mus rau natural compounds [1]. Cov khoom antioxidant ntawm cov khoom xyaw ntuj tuaj yeem pab tiv thaiv ntau yam teeb meem ntawm daim tawv nqaij los ntawm oxidative kev nyuaj siab thiab kev laus [2,3]. Kev laus ntawm daim tawv nqaij tuaj yeem raug ntxias los ntawm ob qho tib si sab hauv (xws li mob lossis telomere shortening) thiab extrinsic (ib puag ncig) yam [4]. Kev laus ntawm daim tawv nqaij ua rau poob ntawm cov laus collagen thiab kev hloov pauv ntawm extracellular matrix (ECM) uas cuam tshuam qhov cuam tshuam kev ua haujlwm, uas ua rau pom qhov qhuav thiab raug rau cov neeg ua phem rau sab nraud, ua rau muaj kev pheej hmoo ntawm daim tawv nqaij mob [5]. Cov txheej txheem no tuaj yeem ua kom nrawm los ntawm ntau cov enzymes, xws li elastases, matrix metalloproteinases (MMPs), thiab hyaluronidases uas tuaj yeem ua rau ECM degradation [6], lossis txawm tias los ntawm kev sib sau ntawm cov kab mob cessive reactive oxygen (ROS) uas tuaj yeem cuam tshuam qhov kev ua haujlwm ntawm tes. [7]. Ib puag ncig yam tseem ceeb, xws li raug UV hluav taws xob, ua rau muaj ntau qhov ntau ntawm ROS uas ua rau tib lub cev thiab cov lus teb ntawm tes raws li kev laus hauv nruab nrab, tab sis nrog cov teebmeem loj. Qhov tseem ceeb, ROS tuaj yeem ua kom muaj zog ntawm cov enzymes ntsig txog kev laus ntawm daim tawv nqaij lossis cov txheej txheem ntawm daim tawv nqaij pigmentation [8,9], thiab yog li muaj cov antioxidants n plays lub luag haujlwm tseem ceeb hauv kev ua kom zoo nkauj.
Hauv xyoo 2018, kev noj ntses thoob ntiaj teb tau kwv yees los ntawm FAO kom sawv ntawm 20.5 kg ib pita [10], uas ua rau muaj ntau ntawm cov khoom lag luam, feem ntau ntawm daim tawv nqaij, thiab pob txha. Cov khoom seem tsim tawm sib raug rau 20-75 feem pua (w/w) ntawm tag nrho cov ntses ntses nyhav, uas ua rau muaj teeb meem ib puag ncig [11,12]. Txawm li cas los xij, cov seem no tseem muaj cov lipids tseem ceeb, cov proteins, thiab cov zaub mov thiab yuav tsum tau muaj txiaj ntsig zoo. Nyob rau hauv xyoo tas los no, cov extracts los ntawm cov pov tseg tsim los ntawm cov ntses kev lag luam tau pom muaj zog xws li antihypertensive, antioxidative, antimicrobial, neuroprotective, antihyperglycemic, anti-aging, thiab anti-inflammatory [13-19]. Atlantic codfish (Gadus rhea) thiab sardine (Sardina pilchardus) yog cov ntses noj ntau tshaj plaws hauv Portugal thiab cov khoom siv tau los ntawm nws cov residues tau pom tias muaj peev xwm nutraceutical zoo, xws li antioxidant, antiproliferative lossis anti-inflammatory kev ua [11,20-22]. Txawm li cas los xij, txij li kev ploitation thiab valorization ntawm cov ntses kev lag luam pov tseg tseem nyob rau theem pib, muaj ntau chav los tshawb nrhiav cov hauv kev rau kev lag luam los hloov cov pov tseg no rau hauv cov khoom lag luam muaj txiaj ntsig zoo, suav nrog cov khoom xyaw kom zoo nkauj.
Hauv kev ua haujlwm dhau los, peb tau tshawb nrhiav kev siv cov thev naus laus zis siab (supercritical CO2 thiab subcritical dej), txhawm rau cais cov seem bioactive los ntawm sardine pov tseg thiab codfish thav ntawv nrog cov txiaj ntsig kev noj qab haus huv [11,22]. Rau cov khib nyiab sardine, peb tau pom tias los ntawm kev thov thawj kauj ruam nrog cov pa roj carbon supercritical (ScCO2) (kom tshem tawm cov lipid feem) ua raws li cov txheej txheem rho tawm nrog cov dej subcritical (SW) nws muaj peev xwm tau txais cov protein hydrolysates nrog cov muaj peev xwm antioxidant thiab cov nyhuv antiproliferative hauv kab mob qog noj ntshav hauv plab [22] Subcritical water extraction/hydrolysis peb kuj tau thov kom tau cov proteins-, peptides- thiab amino acid enrichedxtracts los ntawm codfish thav ntawv thiab peb pom tau hais tias qis ua hauj lwm kub (90 ◦C) nyiam cov extraction ntawm cov tebchaw nrog anti-inflammatory muaj peev xwm nyob rau hauv tib neeg txoj hnyuv epithelial cell. qauv [11]. Feem ntau ntawm cov proteins tam sim no nyob rau hauv codfish thav duab extracts yog collagen thiab collagen fragments. Lwm cov ntsiab lus suav nrog me me ntawm lipids, tshauv, thiab qee cov suab thaj. Sardine extracts tau nplua nuj nyob rau hauv peptides thiab amino acids, thiab lipids, tshauv, thiab suab thaj kuj muaj. Txij li cov ntses-derived proteins thiab peptides yuav dhau los ua ib qho chaw tseem ceeb rau kev lag luam tshuaj pleev ib ce, txoj kev tshawb no tsom mus rau kev soj ntsuam ntxiv cov peev xwm ntawm cov extracts tau los ntawm cov ntses ua cov khoom pov tseg thiab cov khoom siv los ntawm kev ntsuam xyuas ntawm lawv lub peev xwm cosmeceutical [23] . Rau lub hom phiaj no, ntau yam tshuaj, enzymatic, thiab cell-raws li kev soj ntsuam tau siv los tshawb txog cov tshuaj antioxidant, antiageing, anti-hyperpigmentation, anti-inflammatory, thiab antimicrobial teebmeem ntawm cov qauv muab rho tawm. Cov kev tshawb fawb txog kev sib raug zoo kuj tau ua los txheeb xyuas cov ntsiab lus tseem ceeb ntawm cov khoom siv bioactive nrog lub peev xwm cosmetic.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Reagents
3, 4-dihydroxy-l-phenylalanine (L-DOPA), nceb tyrosinase, porcine pancreatic elastase (PPE) hom III, N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN), Tris ({ {11}}amino- 2-hydroxymethyl-propane-1, 3-diol), 2, 20 -azobis (2-}methylpropionamidine) dihydrochloride (AAPH), thiab 20 , 70 -dichlorofluorescein diacetate (DCFH-DA) tau yuav los ntawm Sigma-Aldrich (St. Louis, MO, USA). Calcium-adjusted Mueller Hinton broth (CAMHB) tau yuav los ntawm BD (Sparks, MD, USA). Brain-heart infusion (BHI) tau yuav los ntawm Avantor (Radnor, PA, USA). AnaeroGen ™ Compact hnab tau yuav los ntawm Oxoid (Hampshire, UK). PrestoBlue™, Dulbecco's Modified Eagle Medium (DMEM), Thaum tshav kub kub inactivated Fetal Bovine Serum (FBS), thiab Penicillin-Streptomycin tau txais los ntawm Invitrogen (San Diego, CA, USA). Tib neeg immortalized non-tumorigenic keratinocyte cell kab HaCaT tau txais los ntawm Cell Line Service (Eppelheim, Germany). Tib neeg IL-8 thiab IL-6 Mini TMB ELISA Cov Khoom Siv Txhim Kho tau txais los ntawm Peprotech (London, UK). Tag nrho lwm cov reagents thiab cov kuab tshuaj siv nyob rau hauv txoj kev tshawb fawb tam sim no yog ntawm qib ntsuas thiab yuav los ntawm cov neeg muag khoom muaj.
2.2. Cov qauv
Cov extracts siv nyob rau hauv txoj hauj lwm no yog cov tsim nyob rau hauv peb cov kev tshawb fawb yav dhau los tsom rau cov txheej txheem optimization [11,22]. Luv luv, codfish thav ntawv tau muab los ntawm Pascoal thiab Filhos SA (Gafanha da Nazaré, Portugal) thiab muaj cov ntses qaum thiab cov leeg nqaij. Sardine pov tseg, ua los ntawm taub hau, txha nraub qaum, thiab viscera, tau muab los ntawm Conservas A Poveira SA (Póvoa de Varzim, Portugal). Qhov sib thooj ntawm cov khoom siv raw siv tau nthuav tawm hauv peb cov haujlwm ua ntej [11,22]. Cov protein (47 wt feem pua) thiab tshauv (39 wt feem pua) yog cov khoom tseem ceeb ntawm cov ntses ntses, nrog rau cov lipids thiab carbohydrates tsawg (2 wt feem pua thiab 0.3 wt feem pua, feem). Collagen tau pom tias yog cov protein loj hauv cov ntses ntses, thiab hauv qhov no, nws suav rau ca. 65 feem pua ntawm tag nrho cov ntsiab lus protein ntawm cov pov tseg qub. Nyob rau hauv sib piv, sardine yog ib tug oily ntses, yog li nws cov pov tseg yog ntau nplua nuj nyob rau hauv lipids tshaj codfish thav ntawv. Sardine pov tseg tau pom cov ntsiab lus lipid ntawm 26 wt feem pua thiab cov ntsiab lus ntawm cov protein ntawm 52 wt feem pua, cov seem yog tshauv (17 wt feem pua) thiab carbohydrates (3 wt feem pua).
Cov khoom rho tawm los ntawm codfish thav ntawv (Cf1, Cf2, Cf3, thiab Cf4) thiab sardine pov tseg (S1, S2, thiab S3) xaiv rau txoj haujlwm no tau txais los ntawm cov cuab yeej siv siab hauv lub tshuab ntsuas ntsuas raws li tau piav qhia yav dhau los [11,22 ,24] siv cov xwm txheej uas tau sau tseg hauv Table 1. luv luv, 60 g ntawm av codfish thav ntawv los yog sardine pov tseg (defatted los yog non-defatted) tau thauj mus rau hauv lub tshuab hluav taws xob siab uas tau muab tso rau hauv qhov cub. Lub twj tso kua dej tau hloov pauv ntawm qhov xav tau ntws (ca. 10 mL / min) thiab lub siab tau teem rau 100 bar. Thaum lub siab ncav cuag tus nqi ntawd, lub qhov cub hluav taws xob tau hloov mus, thiab qhov kev sim pib. Cov khoom sib txawv tau sau rau 30 min ntawm qhov kub sib txawv (90-250 ◦C). S1 thiab S3 extracts tau txais tom qab ib tug defatting txheej txheem ntawm sardine pov tseg los ntawm ScCO2 ua ntej SW extraction. Subcritical dej extraction sim tau duplicated. Rau txhua tus qauv muab rho tawm, 25 mL tau muab coj los ua triplicate, lyophilized, thiab hnyav los xam cov txiaj ntsig zoo sib xws. Cov ntaub ntawv txheeb xyuas-cov ntsiab lus protein-yog qhia raws li qhov txhais tau tias ± tus qauv sib txawv (SD) ntawm triplicates. Cov ntaub ntawv hais txog tus yam ntxwv ntawm cov extracts nyob rau hauv cov ntsiab lus ntawm cov ntsiab lus ntawm cov protein, amino acid profile, cov ntxhia hauv cov ntsiab lus tseem ceeb, los yog cov tshuaj lom thiab hnyav hlau tau piav qhia hauv peb cov haujlwm dhau los [11,22].
Cov kev daws teeb meem ntawm Cf1, Cf2, Cf3, Cf4, thiab S2 tau npaj hauv Milli-Q H2O ntawm qhov concentration ntawm 100 mg / mL. Lwm cov qauv, xws li S1 thiab S3, tau yaj hauv DMSO (300 thiab 550 mg / mL, feem) vim lawv cov dej solubility qis dua. Cov qauv tau khov thiab khaws cia ntawm -20 ◦C kom txog thaum siv ntxiv. Rau kev ntsuam xyuas ntawm tes, cov qauv no tau ua kom tsis muaj menyuam los ntawm tshav kub (121 ◦C, 15 min) hauv ib qho autoclave (Tuttnauer 3870 el, Breda, Netherlands).
2.3. Oxygen Radical Absorbance Capacity (ORAC) Kev Ntsuas
ORAC kev soj ntsuam tau ua los ntsuas qhov muaj peev xwm antioxidant ntawm cov qauv ntawm peroxyl radicals (ROO•), raws li txoj kev tsim los ntawm Huang li al. [25], nrog qee qhov kev hloov kho raws li tau tshaj tawm yav dhau los [26]. Luv luv, hauv dub 96-zoo microplate, 150 µL disodium fluorescein (0.3 µM) tau ntxiv rau 25 µL ntawm cov qauv dilutions thiab incubated rau 10 min ntawm 37 ◦C. Tom qab ntawd, cov tshuaj tiv thaiv tau pib los ntawm kev sib ntxiv ntawm 25 µL ntawm 2, 20 - Azobis (2-amidinopropane) dihydrochloride (AAPH, 153 mM), thiab fluorescence (Ex/Em 485 ± 20/528 ± 20 nm) tau ntsuas rau 40 min ntawm 37 ◦C hauv FLx800 fluorescence microplate nyeem ntawv (FL800 Bio-Tek Instruments, Winooski, VT, USA). Tus qauv nkhaus tau npaj siv 5, 10, 20, 30, thiab 40 µM ntawm (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox )). Tag nrho cov kev daws teeb meem tau npaj hauv phosphate-buffered saline (PBS), 75 mM, pH 7.4. Cov txiaj ntsig tau qhia tias yog micromoles ntawm Trolox sib npaug ntawm cov tshuaj tua kab mob antioxidant ib gram ntawm cov extract (µ mol TEAC / g extract).
2.4. Kev soj ntsuam Enzymatic
2.4.1. Elastase Inhibition Assay
Qhov kev ntsuam xyuas no tau ua raws li kev ua haujlwm ntawm Wittenauer li al. [27] nrog qee qhov kev hloov kho raws li tau piav qhia yav dhau los [6]. Elastase inhibitory kev ua ub no yog txiav txim los ntawm ib txoj kev spectrophotometric siv porcine pancreatic elastase (PPE) thiab N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN) raws li enzyme-substrate, los ntawm kev saib xyuas kev tso tawm ntawm p-nitroaniline ntawm 41{ {19}} nm. PPE tau yaj hauv 100 mM Tris (2-amino-2-hydroxymethyl-propane-1,3-}diol)-HCl buffer (pH=8.0) mus rau qhov concentration ntawm 1 mg / mL thiab khaws cia ntawm 20 ◦C hauv aliquots. Nyob rau hnub ntawm kev soj ntsuam, ib qho aliquot tau muab coj los diluted nyob rau hauv qhov tsis rau qhov concentration ntawm 0.03 U / mL, 10 µL tau ntim rau hauv lub qhov dej ntawm daim hlau microtiter ua ke nrog 100 µL ntawm Tris-HCl tsis, thiab 30 µL. ntawm txhua tus qauv. Tom qab 20 min ntawm pre-incubation ntawm 25 ◦C, cov tshuaj tiv thaiv tau pib los ntawm kev ntxiv ntawm 40 µL ntawm substrate AAAPVN (0.55 mM). Kev nqus tau ntsuas rau 20 feeb tom qab ntxiv ntawm AAAPVN ntawm BioTek Instruments EPOCH 2 spectrophotometer microplate nyeem ntawv. Cov kev suav tau ua raws li tau piav qhia hauv Kev Sib Tw (1), qhov twg A tswj thiab Asample sawv cev rau qhov nqus ntawm 410 nm hauv qhov tsis muaj lossis muaj cov qauv, raws li. Txij li thaum DMSO tau siv los yaj cov qauv S1 thiab S3, cov kuab tshuaj no kuj tau sim thiab siv los tswj cov qauv no. Lub peev xwm ntawm cov extracts los inhibit elastase tau soj ntsuam nrog kev nce ntxiv, txhawm rau txiav txim siab kev sib raug zoo ntawm koob tshuaj thiab tsim kom muaj ib nrab-qhov siab tshaj plaws inhibitory concentrations (IC50), qhia txog lub peev xwm ntawm txhua tus qauv hauv kev ua haujlwm enzymatic inhibition rau ib qho ntawm 50 feem pua.
Tag nrho cov txiaj ntsig tau nthuav tawm raws li IC50 txhais tau tias qhov tseem ceeb nrog qis thiab sab sauv txwv ntawm 95 feem pua ntawm kev ntseeg siab, tau los ntawm tsawg kawg peb qhov kev sim ywj pheej.
2.4.2. Tyrosinase Inhibition Assay
Lub tyrosinase inhibitory peev xwm ntawm cov extracts tau soj ntsuam spectrophotometrically siv nceb tyrosinase thiab L-DOPA ua lub substrate [28]. Tyrosinase converts L-DOPA rau dopaquinone, uas yuav ua rau lub voj voog mus ua dopachrome. Kev tsim dopachrome tuaj yeem pom los ntawm kev ntsuas ntawm qhov nqus ntawm 475 nm. Lub substrate tau ntxiv rau cov enzyme nyob rau hauv lub xub ntiag ntawm cov qauv dilutions, mus rau qhov kawg concentration ntawm 30 U/mL tyrosinase thiab 2.5 mM L-DOPA. Txij li thaum DMSO tau siv los yaj cov qauv S1 thiab S3, cov kuab tshuaj no kuj tau sim thiab siv los tswj cov qauv no. Tom qab incubation ntawm 37 ◦C rau 30 min, absorbance yog ntsuas ntawm 475 nm ntawm BioTek Instruments EPOCH 2 microplate spectrophotometer. Tag nrho cov reagents tau npaj nyob rau hauv sodium phosphate buffer (SPB; 0.1 M, pH 6.8), npaj los ntawm kev sib xyaw sodium phosphate dibasic dihydrate thiab sodium phosphate monobasic monohydrate, thiab cov kev suav tau ua raws li tau piav qhia nyob rau hauv Equation (1). Tag nrho cov txiaj ntsig tau nthuav tawm raws li IC50 txhais tau tias qhov tseem ceeb nrog qis thiab sab sauv txwv ntawm 95 feem pua ntawm kev ntseeg siab, tau los ntawm tsawg kawg peb qhov kev sim ywj pheej.
2.5. Antimicrobial Susceptibility Testing
Lub hom phiaj ntawm cov kab mob uas tau xaiv rau cov tshuaj tua kab mob yog cov kab mob gram-negative Klebsiella pneumoniae CECT 8453 thiab cov kab mob gram-positive Staphylococcus aureus ATCC 6538 thiab Cutibacterium acnes ATCC 6919T. Rau K. pneumoniae CECT 8453 thiab S. aureus ATCC 6538, kev soj ntsuam tau ua raws li txoj kev ua kua dej microdilution ntawm CLSI M07-A10 cov lus qhia raws li yav dhau los tau piav qhia los ntawm Rodrigues li al. [29]. Nyob rau hauv luv luv, extract cov ntsiab lus tau muab faib nyob rau hauv ib puag ncig hauv qab microtiter 96-zoo phaj thiab 2-fold serially diluted nyob rau hauv calcium-hloov Mueller Hinton broth (CAMHB; BD, Sparks, MD, USA) kom tau txais ib concentration ntau yam kev daws teeb meem. Lub inoculum tau npaj siv txoj kev loj hlob kom ua tiav homogenous suspension nyob rau hauv cov kua ntsev. Lub inoculum kho tau ntxiv diluted hauv CAMHB los lav tias, tom qab inoculation, txhua qhov dej muaj nyob ib ncig ntawm 5 × 104 CFU. Cov paib tau incubated nyob rau hauv aerobic tej yam kev mob ntawm 37 ◦C rau 16 mus rau 20 teev. Rau C. pob txuv, qhov kev ntsuam xyuas tau ua raws li tau piav qhia yav dhau los nrog kev siv lub hlwb-lub plawv infusion (BHI) (Avantor, Radnor, PA, USA) kua zaub es tsis txhob CAMHB thiab los ntawm incubating lub microtiter daim hlau rau 70–74 h ntawm 37 ◦ C nyob rau hauv anaerobic jars uas muaj cov huab cua tiam system AnaeroGen ™ Compact (Oxoid, Hampshire, UK).
Rau txhua qhov kev daws teeb meem tshuaj ntsuam xyuas, kev tswj zoo (CAMHB lossis BHI thiab diluted inoculum), kev tswj tsis tau nruab nrab (uninoculated CAMHB lossis BHI), thiab kev tswj kom tsis muaj menyuam (uninoculated 2-fold extract stock solution hauv CAMHB lossis BHI) tau ua raws li. Yam tsawg kawg nkaus inhibitory concentration (MIC) qhov tseem ceeb yog qhov qis tshaj ntawm cov qauv uas pom tau tias inhibited microbial kev loj hlob tom qab incubation. Thaum xav tau, MIC qhov tseem ceeb tau lees paub tias siv lub cell viability reagent PrestoBlue™ (Invitrogen, San Diego, CA USA) ua raws li cov chaw tsim khoom cov lus qhia. Ib qho ntxiv MIC tus nqi, tsim MIC *, kuj tau tsim thiab txhais tias yog qhov qis tshaj plaws ntawm cov qauv uas cov kab mob loj hlob tau pom thiab cuam tshuam sib txawv hauv kev sib piv rau kev tswj hwm zoo. Yam tsawg kawg nkaus bactericidal concentration (MBC) qhov tseem ceeb tau tshaj tawm raws li qhov qis tshaj ntawm cov qauv ua rau tsawg kawg yog 99.9 feem pua txo qis hauv cov kab mob uas muaj peev xwm suav nrog piv rau qhov pib inoculum thiab rau lub sijhawm sib npaug. Cov txiaj ntsig tau nthuav tawm raws li qhov nruab nrab ntawm cov txiaj ntsig tau txais tom qab peb cov khoom siv roj ntsha. Txij li thaum DMSO tau siv los yaj cov qauv S1 thiab S3, cov kuab tshuaj no kuj tau sim los xyuas kom meej tias qhov kawg concentration siv tsis cuam tshuam rau lub hom phiaj kab mob, yog li kev soj ntsuam.
2.6. Cell-based Assays
2.6.1. Cell Culture
Tib neeg keratinocyte cell kab HaCaT (CLS, Lub teb chaws Yelemees) yog kab lis kev cai hauv tus qauv Dulbecco's Modified Eagle Medium (DMEM) ntxiv nrog 10 feem pua (v/v) fetal bovine serum (FBS) thiab 1 feem pua (v/v) penicillin-streptomycin. Cov hlwb tau niaj hnub khaws cia ua monolayers nyob rau hauv 75 cm2 kab lis kev cai flasks thiab incubated ntawm 37 ◦C nrog 5 feem pua CO2 nyob rau hauv ib tug humidified huab cua.
2.6.2. Hauv Vitro Cytotoxicity
Kev soj ntsuam cytotoxicity tau ua raws li kev ua haujlwm dhau los [6]. Luv luv, HaCaT hlwb tau noob ntawm qhov ntom ntawm 1.4 × 105 hlwb / cm2 hauv 96 lub phaj zoo. Tom qab 3 hnub, cov cell raug incubated nrog sib txawv concentration ntawm txhua tus qauv (Cf1; Cf2; Cf3; Cf4; S2—50, 25, 12.5, 6.25, 3.13, 1.56, {{30} }.78, 0.39; S1—3, 1.5, 0.75, 0.38, {{40}}.19, {{51} } }. 0.09, 0.04 mg / mL) diluted hauv kab lis kev cai nruab nrab (DMEM nruab nrab muaj 0.5 feem pua FBS). Lub qhov dej uas muaj cov hlwb incubated nkaus xwb nrog kab lis kev cai nruab nrab ntxiv nrog 0.5 feem pua (v/v) ntawm FBS tau siv los tswj. Kev tswj cov kuab tshuaj nrog 50 feem pua dej lossis 1 feem pua DMSO hauv kab lis kev cai nruab nrab kuj tau ua kom tsis suav cov kuab tshuaj lom. Tom qab 24 teev ntawm incubation, lub cell viability raug soj ntsuam siv PrestoBlue® (5 feem pua v/v nyob rau hauv kab lis kev cai nruab nrab) rau 2 h ntawm 37 ◦C, 5 feem pua CO2, raws li cov chaw tsim tshuaj paus cov lus qhia. Tom qab no, cov fluorescence ntawm txhua qhov dej tau ntsuas (Ex./Em. 560 ± 20/590 ± 20 nm) hauv FLx800 fluorescence microplate nyeem ntawv (BioTek Instruments, Winooski, VT, USA). Cell viability tau qhia raws li qhov feem pua ntawm cov cell siv tau txheeb ze rau kev tswj hwm. Peb qhov kev sim ywj pheej tau ua hauv triplicate.
2.6.3. Cellular Antioxidant Kev Ua Haujlwm
Cellular antioxidant kev ua haujlwm tau raug soj ntsuam raws li tau piav qhia yav dhau los [6,30], nrog qee qhov kev hloov kho. Luv luv, HaCaT hlwb tau noob ntawm qhov ntom ntawm 1.4 × 105 hlwb / cm2 hauv 96 daim hlau zoo thiab kev tsim ntawm intracellular ROS tau saib xyuas siv 20,70 -dichlorofluorescein diacetate ( DCFH-DA) raws li kev sojntsuam fluorescent. 72 h tom qab cov noob, cov hlwb raug ntxuav nrog PBS thiab incubated nrog cov tsis muaj tshuaj lom ntau ntawm cov qauv (0.1875 mg / mL; 0.375 mg / mL; 0.75 mg / mL) ntxiv rau 25 µM DCFH-DA hauv PBS rau 1 h. Tom qab ntawd, cov hlwb raug ntxuav dua nrog PBS thiab incubated nrog cov kev ntxhov siab inducer (600 µM AAPH hauv PBS) rau 1 h. Tom qab ntawd, fluorescence raug ntsuas hauv FL800 microplate fluorescence nyeem ntawv (Bio-Tek Instruments, Winooski, VT, USA) (Ex/Em 485 ± 20/528 ± 20 nm). Cov txiaj ntsig tau qhia raws li ROS feem pua ntawm cov kev tswj tsis tau kho (cov cell kho nrog DCFH-DA thiab AAPH). Peb qhov kev sim ywj pheej tau ua hauv triplicate.
2.6.4 ib. Kev ntsuam xyuas ntawm IL-6 thiab IL-8 Secretion
Kev sim tau ua raws li tau piav qhia yav dhau los [31], nrog rau ntau qhov kev hloov kho. Luv luv, HaCaT hlwb tau noob ntawm qhov ntom ntawm 1 × 105 hlwb / cm2 hauv 12 qhov zoo daim hlau. Tom qab 3 hnub, cov hlwb raug txhawb nrog 15 µg / mL ntawm lipopolysaccharides (LPS) los ntawm Escherichia coli thiab co-incubated nrog peb qhov sib txawv ntawm txhua qhov extract ({{10}}}.1875 mg/mL; {{ 14}}.375 mg/mL; 0.75 mg/mL) diluted hauv kab lis kev cai nruab nrab (DMEM nruab nrab uas muaj 0.5 feem pua FBS). Cells incubated tsuas yog nrog LPS thiab cov hlwb incubated nrog cov kab lis kev cai tsuas yog siv los tswj qhov zoo thiab tsis zoo, raws li. Tom qab 24 teev, supernatants tau sau, centrifuged rau 10 min ntawm 2000 g, thiab khaws cia ntawm −80 ◦C kom txog rau thaum kev soj ntsuam ntxiv. IL-6 thiab IL-8 qib tau ntsuas los ntawm enzyme-linked immunosorbent assay (ELISA), siv cov khoom siv muag khoom (PeproTech; London, UK), raws li cov chaw tsim khoom cov lus qhia, nrog absorbance ntsuas ntawm 450 nm nrog wavelength kho teeb tsa ntawm 620 nm hauv microplate spectrophotometer (EPOCH 2, BioTek Instruments, Winooski, VT, USA). Cov txiaj ntsig tau qhia raws li IL-6 lossis IL-8 feem pua txheeb ze rau kev tswj hwm zoo (cov cell txhawb nqa nrog LPS). Peb qhov kev sim ywj pheej tau ua hauv triplicate.
2.7. Kev txheeb cais
ORAC thiab cell-based assay cov txiaj ntsig tau qhia raws li tus nqi nruab nrab ± SD, tau los ntawm tsawg kawg peb qhov kev sim ywj pheej. Rau qhov kev ntsuam xyuas enzymatic thiab cytotoxicity, IC50 qhov tseem ceeb tau txiav txim siab los ntawm cov koob tshuaj tiv thaiv kab mob los ntawm cov phiaj xwm log10 siv GraphPad Prism 8.4.3. software (GraphPad Software, Inc., La Jolla, CA, USA). Cov txiaj ntsig tau nthuav tawm raws li IC50 nrog 95 feem pua ntawm kev ntseeg siab. Kev txheeb xyuas cov txiaj ntsig tau ua los ntawm cov software qub. Thaum homogeneous variance thiab ib qho kev faib tawm ntawm cov ntaub ntawv raug txheeb xyuas, cov txiaj ntsig tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA), tom qab ntawd los ntawm Tukey test rau ntau qhov sib piv. Nyob rau hauv cov ntaub ntawv ntawm heterogeneous variances los yog yog hais tias cov ntaub ntawv tsis yog ib txwm muab faib, ib tug tsim nyog unpaired Student's t-test tau ua los txiav txim seb cov txhais tau tias txawv heev. Ib qho p-tus nqi Tsawg dua lossis sib npaug rau 0.05 tau txais raws li qhov tseem ceeb hauv txhua qhov xwm txheej. Antimicrobial susceptibility test results yog qhia raws li tus nqi nruab nrab, tau los ntawm tsawg kawg peb qhov kev sim ywj pheej.
3. Cov txiaj ntsig thiab kev sib tham
This study aims to investigate the cosmeceutical potential of protein-rich extracts that were produced by high-pressure technologies from fishery industry wastes, namely sardine wastes and codfish frames [11,22]. The selection of extracts was based on previous results regarding their characterization and process conditions. For codfish, all extracts were selected aiming at evaluating the impact of the extraction temperature on the recovery of compounds with promising bioactive effects on the skin. For sardine extracts, only three extracts derived from both defatted and non-defatted raw materials, processed at higher temperatures (190 and 250 ◦C) and with the highest extraction yield (>45.7 g / 100 g) raug xaiv. Cov txiaj ntsig hais txog tag nrho cov ntsiab lus protein ntawm txhua qhov extract tau nthuav tawm hauv Table 1.
3.1. Antioxidant, Anti-Ageing, thiab Anti-Hyperpigmentation Activities
Lub peev xwm cosmeceutical nyhuv ntawm cov extracts tau pib tshuaj xyuas siv tshuaj thiab cov tshuaj enzymatic los soj ntsuam lawv cov tshuaj antioxidant, tiv thaiv kev laus, thiab tiv thaiv hyperpigmentation teebmeem. Rau qhov muaj peev xwm antioxidant, ORAC kev soj ntsuam tau raug xaiv raws li nws ntsuas lub peev xwm ntawm cov qauv kom tshem tawm cov kab mob lom ROS, uas yog peroxyl radicals, uas yog suav tias yog ib qho tseem ceeb inducers ntawm daim tawv nqaij laus [2,32]. Cov nyhuv tiv thaiv kev laus kuj raug soj ntsuam los ntawm elastase inhibition vim tias qhov enzyme no tau tshaj tawm tias yog lub luag haujlwm rau kev degradation ntawm elastin thiab lwm yam ECM proteins [6]. Rau cov nyhuv anti-hyperpigmentation, tyrosinase assay tau siv, los ntsuas lub peev xwm ntawm cov qauv los inhibit melanin ntau lawm. Table 2 piav qhia txog ORAC thiab IC50 qhov tseem ceeb ntawm txhua qhov kev rho tawm.
Peb cov txiaj ntsig tau pom tias sardine thiab codfish extracts nthuav tawm cov haujlwm antioxidant thiab inhibition cuam tshuam ntawm elastase thiab tyrosinase enzymes 'kev ua haujlwm. Ntawm cov qauv sardine, S1 pom qhov siab tshaj ORAC tus nqi (1.94 ± 0.08 µmol TEAC/mg extract) ua raws li S3 thiab S2. Cov txiaj ntsig no yog los ntawm kev soj ntsuam antioxidant yav dhau los los ntawm lwm txoj hauv kev (2,2-diphenyl-1-picrylhydrazyl-DPPH assay) qhov twg cov extract S1 tau qhia qhov qis tshaj IC50 qhov tseem ceeb [22]. Lub peev xwm ntau dua ntawm cov tshuaj peroxyl radicals ntawm cov qauv S1 tuaj yeem muab tau los ntawm peptides nrog cov amino acid sib txawv nyob rau hauv cov extracts txij li kev sib cuam tshuam ntawm lawv tuaj yeem cuam tshuam lub peev xwm radical scavenging [33]. Tsis tas li ntawd, cov tebchaw tsim tawm hauv Maillard lossis lwm cov tshuaj tiv thaiv thermo-oxidation tuaj yeem cuam tshuam rau kev ua haujlwm antioxidant ntawm cov qauv [34]. Txawm hais tias tus nqi qis tshaj ORAC, cov qauv S2 thiab S3 yog cov uas muaj peev xwm siab tshaj plaws hauv inhibiting tyrosinase thiab elastase cov dej num, feem.
Ntawm cov ntses ntses, Cf4 tau pom tias muaj cov tshuaj tiv thaiv kab mob siab tshaj plaws thiab tiv thaiv hyperpigmentation. SEC-GPC tsom xam ntawm cov extracts tau pom tias peptides ntawm qhov hnyav molecular tau txais thaum nce qhov kub ntawm cov extraction mus txog 250 ◦C [11]. Lub peev xwm elastase inhibition ntawm tus qauv no yog nyob rau hauv qhov ntau ntawm cov txiaj ntsig pom rau lwm cov Cf rho tawm, xws li Cf1 thiab Cf3. Txawm li cas los xij, rau qhov kev sim siab, ob qhov kev rho tawm no tsis pom muaj kev cuam tshuam rau tyrosinase.
Nyob rau hauv cov ntaub ntawv, qee cov ntaub ntawv qhia tau hais tias extracts los ntawm marine by-products tam sim no muaj feem xyuam rau antioxidant kev ua si thiab inhibition ntawm tyrosinase. Piv txwv li, cov extracts muab tau los ntawm marine (Scophthalmus maximus) los ntawm cov khoom lag luam los ntawm alkaline hydrolysis, tau ua kom pom cov tshuaj tiv thaiv antioxidant uas tau nkag los ntawm cov tshuaj sib txawv: 1, 1-diphenyl-2- picrylhydrazyl (DPPH) radical-scavenging peev xwm ( 36.12 feem pua ntawm kev tswj), ABTS (2,20 -azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)) txoj kev (12.81 µg BHT/mL) thiab crocin bleaching assay (8. {28}}3 µg Trolox/mL) [35]. Hauv lwm txoj kev tshawb fawb, enzymatic rho tawm ntawm alum-salted jellyfish (Lobonema smithii) tau pom tias tsim cov hydrolysates nrog cov tshuaj antioxidant siab (IC{{20}}}. 9 mg/mL rau ABTS e DPPH assays) thiab tyrosinase inhibitory muaj peev xwm, nrog IC50 qhov tseem ceeb ntawm 14.1 thiab 24.5 mg / mL [36], uas yog nyob rau hauv tib qhov kev txiav txim ntawm qhov loj npaum li cov tau txais hauv txoj haujlwm no. . Tsis tas li ntawd, cov extracts nrog cov txiaj ntsig zoo sib xws (ORAC qhov tseem ceeb - 0.4 mus rau 3.5 µmol TEAC/mg extract) tau los ntawm lwm hom khoom noj khoom haus residues, uas yog winemaking khib nyiab [6]. Txawm li cas los xij, cov dej cawv residues tau nthuav tawm ntau dua tyrosinase (IC50 los ntawm 4.0 txog 0.14 mg extract / mL) thiab elastase (IC50 los ntawm 3.4 txog 0.1 mg extract / mL) inhibitory muaj peev xwm dua li cov tau txais hauv txoj haujlwm no, tej zaum vim muaj cov phenolic tebchaw. uas tau lees paub tias muaj ntau yam bioactivities. Nws yog ib qho tseem ceeb uas yuav tsum tau hais tias hauv peb txoj kev tshawb fawb peb tau siv cov nceb tyrosinase los tshuaj xyuas cov nyhuv anti-hyperpigmentation ntawm cov extracts, vim tias cov enzyme no tau siv dav hauv kev ntsuam xyuas ntau dhau [37]. Txawm li cas los xij, txij li muaj qee qhov kev tsis sib haum xeeb txog qhov zoo sib xws thiab homology ntawm cov enzyme no nrog cov tsiaj txhu / tib neeg tyrosinase [38-40], cov kev tshawb fawb yav tom ntej uas cuam tshuam nrog cov kab mob ntawm tes yuav tsum tau txiav txim siab los ntsuas qhov muaj peev xwm tiv thaiv hyperpigmentation ntawm sardine thiab codfish extracts.
Txhawm rau txheeb xyuas cov tebchaw twg tuaj yeem ua lub luag haujlwm rau cov lus teb bioactive ntawm cov extracts, kev sib raug zoo ntawm cov ntaub ntawv bioactivity thiab cov extracts ' amino acid muaj pes tsawg leeg tau qhia yav dhau los [11,22] tau ua (Table S1). Rau kev ua haujlwm antioxidant, qhov sib raug zoo tshaj plaws (R2 Ntau dua lossis sib npaug ntawm 0.7) tau txais ntawm ORAC tus nqi thiab tag nrho threonine, dawb valine, nrog rau dawb thiab tag nrho leucine rau tag nrho cov extracts. Nyob rau hauv cov ntaub ntawv ntawm cov qauv sardine, muaj kev sib raug zoo (R2 Ntau dua lossis sib npaug rau 0.94) kuj tau txais dawb thiab tag nrho cov ntsiab lus tryptophan. Raws li, tag nrho cov amino acids yav dhau los tau tshaj tawm tias muaj cov khoom antioxidant hauv ntau lub qauv [41–43]. Rau elastase thiab tyrosinase inhibition kev ua ub no, lub siab tshaj plaws correlation coefficients (R2 Ntau tshaj los yog sib npaug rau 0.8) tau txais rau tag nrho cov protein cov ntsiab lus thiab dawb arginine, raws li, qhia tias cov tebchaw yuav muaj lub luag hauj lwm tseem ceeb nyob rau hauv lub peev xwm. anti-aging thiab anti-hyperpigmentation nyhuv ntawm ntses kev lag luam pov tseg kwj extracts.
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