Ntsuam Xyuas Cov Antioxidants, Whitening Thiab Antiaging Properties Ntawm Rice Protein Hydrolysates
Mar 27, 2022
Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791
Hui-Ju Chen1,2, Fan-Jhen Dai 2, Cheng-You Chen 3, Siao-Ling Fan 2, Ji-Hong Zheng 4, Yu-Chun Huang 2, Chi-Fai Chau 1, Yung-Sheng Lin3,4, 5,* thiab Chin-Shuh Chen1,*
Department of Food Science thiab Biotechnology, National Chung Hsing University, Taichung 402204, Taiwan; d103043004@mail.nchu.edu.tw (H.-JC); chaucf@nchu.edu.tw (C.-FC)
Healthmate Co., Ltd., Changhua City 500016, Taiwan; jane@healthmate.com.tw (F.-JD); eileen@healthmate.com.tw (S.-LF); tina@healthmate.com.tw (Y.-CH)
Ph.D. Program hauv Khoom Siv thiab Tshuaj Engineering, National United University, Miaoli 360001, Taiwan; D0612002@smail.nuu.edu.tw
Department of Chemical Engineering, National United University, Miaoli 360001, Taiwan; U0714049@smail.nuu.edu.tw
Lub koom haum ntawm Food Safety and Health Risk Assessment, National Yang-Ming University, Taipei 112304, Taiwan Correspondence: linys@nuu.edu.tw (Y.-SL); cschen@mail.nchu.edu.tw (C.-SC)
Lus Nug: Cov nroj tsuag tau txais cov protein hydrolysates muaj peev xwm siv tau hauv kev noj zaub mov. Rice protein hydrolysates (RPHs), uas yog ib qho zoo heev ntawm cov proteins, tau nyiam cov xim rau kev loj hlob ntawm cosmeceuticals. Txawm li cas los xij, ob peb txoj kev tshawb fawb tau tshaj tawm tias muaj peev xwm siv RPH hauv kev tshuaj xyuas, thiab qhov kev tshawb fawb no tau tshuaj xyuas lawvantioxidantkev ua ub no thiab inhibitory kev ua ub no ntawm daim tawv nqaij laus enzymes. Cov txiaj ntsig tau qhia tias tag nrho cov phenolic thiab avonoid concentrations yog 2.06 士 0.13 mg gallic acid sib npaug / g RPHs thiab 25.96 士 0.52 µg quercetin sib npaug / g RPHs , raws. RPHs tau qhia txog kev ua haujlwm ntawm koob tshuaj rau kev tshem tawm cov dawb radicals los ntawm 1,1- diphenyl-2-picrylhydrazyl [half-maximal inhibitory concentration (IC50)=42.58 士 2.1 mg/g RPHs] thiab 2 ,2′ -and-bis (3-ethylbenzothiazoline-6-sulfonic acid) (IC50=2.11 士 0.88 mg/g RPHs), koob tshuaj-dependent reduction peev (6.95 士 1.40 mg vitamin C sib npaug / g RPHs) thiab oxygen radical absorbance peev xwm (473 µmol Trolox sib npaug / g RPHs). Cov concentrations ntawm cov tshuaj RPH yuav tsum tau ua kom tiav 50 feem pua inhibition ntawm hyaluronidase thiabtyrosinaseCov kev ua ub no tau txiav txim siab yog 8.91 thiab 107.6 mg / mL, feem. Txoj kev tshawb no pom tau tias RPHs muajantioxidantantihyaluronidase thiab tshuaj tiv thaiv,tyrosinasekev ua ub no rau kev siv tshuaj pleev ib ce yav tom ntej.
Ntsiab lus: nplej protein hydrolysate; antioxidant; hyaluronidase; tyrosinase; tshuaj pleev ib ce
Cistanche muaj ib qhoantioxidantthiabkho hniav dawbnyhuv
1. Taw qhia
Ultraviolet hluav taws xob raug yog lub luag hauj lwm rau photoaging (los yog extrinsic aging); Nyob rau hauv sib piv, reactive oxygen hom tsim nyob rau hauv cell metabolism thiab deterioration ntawm lom functions yog lub luag hauj lwm rau intrinsic laus [1,2]. Cov zaub mov ua tiav feem ntau muaj ntujantioxidantsxws li catechins, ascorbic acid, tocopherols, rosmarinic acid, thiab phenolic rho tawm los ntawm ntau yam nroj tsuag. Kev tshawb fawb tau ua rau hauv ntujantioxidantstam sim no suav tias yog cov pov thawj uas tsis yog kev cai. Ntuj losantioxidantsmuaj kev ntshaw ntau dua li cov khoom siv tshuajantioxidantstxij li qee cov hluavtaws antioxidants tau tshaj tawm tias yog carcinogenic [3]. Rice (Oryza sativa) yog cov khoom noj khoom haus tseem ceeb rau cov neeg thoob ntiaj teb, tshwj xeeb yog cov neeg nyob hauv Asia. Lub ntiaj teb cov nplej txhua xyoo yog kwv yees li 741 lab tons [4]. Hauv cov tebchaws Esxias, cov nplej tau tshaj tawm tias yog lub hauv paus ntawm 75 feem pua ntawm kev siv zog ntawm ntau tshaj 2 billion tus neeg [5]. Cov nplej ntau lawm ua rau muaj qhov sib npaug ntawm cov khoom lag luam. Cov khoom seem ntawm cov txheej txheem ua mov muaj feem ntau ntawm cov nplej protein (~60–85 feem pua) tab sis raug pov tseg lossis siv rau kev pub tsiaj [6–8]. Peptides tau txais los ntawm ntau yam protein hydrolysates tau tshaj tawm ua qhov muaj peev xwmantioxidants[9]. Natural thiab nontoxic antioxidants tuaj yeem yog li muaj peev xwm muab rho tawm los ntawm cov zaub mov protein hydrolysates. Ntau tus kws tshawb fawb tau ua haujlwm cov qauv lipid-nplua nuj thiab tshaj tawm cov protein hydrolysates nrog rau mis nyuj, zein, thiab kua protein peptides kom muaj qhov tseem ceeb.antioxidantCov yam ntxwv, suav nrog kev tshem tawm cov dawb radicals, inhibition ntawm cov zaub mov, thiab in-vitro lipid peroxidation, thiab chelation ntawm cov hlau hloov pauv [10-12].
Hyaluronic acid (HA) pab kom cov tawv nqaij rejuvenate vim nws tsub kom viscosity, muaj noo noo, thiab ua rau extracellular kua permeable. Vim nws muaj peev xwm tuav tau dej zoo, HA ua rau cov hluas, moisturization, thiab smoothness ntawm daim tawv nqaij thiab txo cov degree ntawm wrinkles [13,14]. Hmoov tsis zoo, qib HA hauv daim tawv nqaij ib txwm txo qis nrog hnub nyoog. Hyaluronidase yog ib qho enzyme uas rhuav tshem HA, ua rau poob ntawm daim tawv nqaij lub zog, elasticity, thiab noo noo, uas nyob rau hauv lem, ua rau tawv nqaij laus. Yog li ntawd, wrinkles tuaj yeem kho los ntawm inhibiting hyaluronidase thiab tswj HA cov ntsiab lus ntawm daim tawv nqaij [15,16]. Lub enzyme tsim melanintyrosinasetxhawb nqa qhov tseem ceeb rau tus nqi-txheej theem ntawm cov txheej txheem los ntawm kev tsim melanin. Yog li ntawd, pigmentation tsis meej yog feem ntau kho thiab daim tawv nqaij brightening tiav los ntawm inhibiting los yog downregulating.tyrosinasekev ua si [17,18].
Hauv ntau qhov kev tshawb fawb, cov nplej protein hydrolysates thiab peptides uas tuaj yeem tau txais los ntawm lawv tau pom tias muajantioxidant, antihypertensive, thiab antitumor kev ua ub no [19,20]. Cov txiaj ntsig zoo rau tib neeg kev noj qab haus huv ntawm cov zaub mov-peptides thiab cov protein tau maj mam raug lees paub [21]. Cov neeg siv khoom xav tau nce ntxiv tias cov kev lag luam tshuaj pleev ib ce thiab kev noj qab haus huv siv cov tshuaj bioactive hauv ntuj. Rice protein hydrolysates (RPHs) tau ntxim nyiam ua ib qho zoo heev ntawm cov proteins. Txawm li cas los xij, qee qhov kev tshawb fawb tau tshaj tawm cov yam ntxwv thiab kev ua haujlwm ntawm RPHs. Yog li ntawd, qhov kev tshawb fawb no tau soj ntsuam covantioxidantkev ua haujlwm thiab hyaluronidase thiabtyrosinase- inhibitory
cov haujlwm ntawm RPHs.
2. Cov txiaj ntsig thiab kev sib tham
2.1. Tag Nrho Phenolic Concentration (TPC) thiab Tag Nrho Cov Ntsiab Lus Flavonoid (TFC)
Tus qauv hauv TPC qhov kev ntsuam xyuas yog gallic acid ntawm ntau qhov ntau. Kev nqus siab dua qhia tau tias TPC siab dua. Lub TPC ntawm cov qauv RPH tau txais los ntawm kev nkag mus rau RPH cov qauv 'optical absorbance qhov tseem ceeb rau hauv lub gallic acid calibration nkhaus. Los ntawm kev npaj qhov RPH concentration tawm tsam phenolic concentration (Daim duab 1a), qhov nruab nrab TPC ntawm 2.06 士 0.13 mg GAE/g RPHs tau txais. A TFC ntawm 25.96 士 0.52 µg QE/g RPHs tau txais los ntawm kev ua raws li cov txheej txheem zoo sib xws (Daim duab 1b). Daim duab 1c ntxiv hais txog TPC thiab TFC ntawm cov qauv RPHs. Nws nthuav tawm tias kev sib raug zoo ntawm TPC thiab TFC tuaj yeem qhia tau tias yog y=0.0121x ntxiv rau 0.0659, qhov twg x thiab y yog TPC thiab TFC, feem.
TPC ntawm RPHs suav nrog cov concentrations ntawm phenolic amino acids thiab phenolic compounds ntawm peptides. Protein-phenolic compound kev sib cuam tshuam feem ntau suav nrog covalent thiab noncovalent bonding. Phenolic compounds raug tso tawm thaum lub sij hawm enzymatic hydrolysis. Cov enzymes tshwj xeeb tuaj yeem ua kom puas cov protein-polyphenol complexes; qhov no ua rau muaj ntau cov phenolic tebchaw thiab peptides nrog phenolic pawg, xws li tyrosine, raug tso tawm [22]. Ib qho kev sib raug zoo tau raug tshaj tawm ntawm tag nrho cov ntsiab lus polyphenol ntawm cov nplej thiab lawv cov haujlwm lom neeg. Polyphenols paub zoo tias muaj zogantioxidantkev ua si [23]. Txawm hais tias pom muaj tsawg dua, terpenes [24] lossis sesquiterpenes [25] hauv nplej kuj tuaj yeem ua rauantioxidantkev ua si.
2.2. Kev ua ntawm Antioxidants
2.2.1. Radical Scavenging Activity ntawm DPPH Dawb Radicals
Daim duab 2 qhia txog DPPH dawb radical scavenging kev ua hauv RPH tov. Qhov siab dua ntawm cov tshuaj tau pom los ua kom muaj kev ua haujlwm siab dua. Ib nrab-qhov siab tshaj plaws inhibitory concentration (IC50), uas yog cov extract concentration uas ib nrab ntawm tag nrho cov DPPH dawb radicals yuav scavenged, yog 42.58 士 2.1 mg / mL ntawm mov peptides.
2.2.2. Kev Tshawb Fawb Kev Ua Haujlwm ntawm ABTS Dawb Radicals
RPHs 'ABTS dawb radical scavenging kev ua ub no, qhia hauv daim duab 3, yog siab dua thaum muaj kev ua haujlwm ntau dua. IC50 yog 2.11 士 0.88 mg/mL ntawm mov peptides. Cov txiaj ntsig no tau qhia tias RPHs muaj zog ABTS dawb radical scavenging kev ua haujlwm. Cov sulfur-muaj cov amino acids, suav nrog Met thiab Cys, thiab hydrophobic amino acids, xws li Ala, Val, Ile, Leu, Met, Cys, Tyr, Phe, Sim thiab Pro, tej zaum yuav yog ib qho tseem ceeb hais txog ABTS dawb radical scavenging. kev ua si [26]. Ntxiv mus, cov amino acids uas muaj ntxhiab, nrog rau Phe thiab Tyr, kuj txhawb ABTS dawb radical scavenging kev ua si vim lawv benzene rings pub protons rau electron tsis muaj radicals [27].
Daim duab 3. Kev cuam tshuam ntawm RPH concentration ntawm qhov muaj peev xwm scavenging ntawm 2,2′ -azino-bis(3-ethylbenz- othiazoline-6-}sulfonic acid) (ABTS).
Hauv txoj kev tshawb no, IC50 tus nqi ntawm ABTS dawb radical scavenging kev ua haujlwm qis dua DPPH dawb radical scavenging kev ua, pom zoo nrog cov txiaj ntsig ntawm Jatropha curcas L. noob plhaub thiab kernel [28] thiab jujube txiv hmab txiv ntoo noob thiab tev pulp [29]. Qhov kev tshawb pom no kuj sib xws nrog rau daim ntawv tshaj tawm ntawm cov nplej bran protein hydrolysates nrog 43.98–66.25 µmol Trolox sib npaug / g qauv thiab 403.28–430.12 µmol Trolox sib npaug / g qauv rau DPPH dawb radical scavenging kev ua ub no thiab ABTS.
Ib qho laj thawj yog vim li cas qhov sib txawv ntawm kev solubility ntawm DPPH dawb radical (roj soluble) thiab ABTS dawb radical (roj / dej soluble) [30,31]. CovantioxidantLub peev xwm ntawm nplej bran protein hydrolysates tau cuam tshuam los ntawm nws qhov hnyav molecular profile, amino acid muaj pes tsawg leeg thiab hydrophobicity [32].
2.2.3. Txo peev xwm
Qhov txo qis peev xwm txheeb xyuas rau RPHs tau nthuav tawm hauv daim duab 4. Lub peev xwm txo tau nce nrog RPHs concentration. Lub peev xwm txo qis yog 6.95 士 1.40 mg VCE / g RPHs, qhia tias RPHs yog qhov ua tau zoo.antioxidant.
Daim duab 4. Kev cuam tshuam ntawm qhov concentration ntawm RPH ntawm kev txo qis.
2.2.4. Oxygen Radical Absorbance Peev Xwm (ORAC)
Qhov kev ntsuam xyuas ORAC muaj qhov zoo dua lwm txoj hauv kev rauantioxidantKev txiav txim siab ua haujlwm, suav nrog cov reactants siv los ua peroxy radicals nrog cov txheej txheem zoo sib xws ntawm cov tshuaj tiv thaiv thiab redox peev xwm rau lub cev oxidants; tag nrho cov nqi thiab protonation xeev uas covantioxidantsreact zoo li cov hauv tib neeg lub cev [33] Txoj kev ORAC kuj tseem muaj kev cuam tshuam txog kev noj qab haus huv rau kev ua tau zoo ntawmantioxidantsnyob rau hauv tib neeg lub cev. Daim duab 5 qhia txog cov txiaj ntsig ntawm ORAC tsom xam ntawm RPHs thiab tus qauv Trolox hauv ntau qhov ntau. Lub ORAC tau muab los ntawm qhov sib npaug regression ntawm lub calibration nkhaus hais txog cov net AUC rau Trolox concentration. Cov txiaj ntsig tau qhia tias RPHs muaj ORAC ntawm 473 µmol TE / g RPHs.
Antioxidantpeptides lossis amino acids tuaj yeem tau txais los ntawm enzymatic protein hydrolysis, uas ua rau muaj zog tiv thaiv oxidants [34]. Cov hlau ion chelation, lipid peroxidation inhibition, thiab dawb radical scavenging ntawm biologically active peptides yog lub luag haujlwm rau lawv cov haujlwm antioxidant. Dawb radicals tuaj yeem quenched thiab qhia txog oxidative-kev nyuaj siab-txo cov proteins thiab enzymes upregulated los ntawmantioxidantpeptides. Cov tshuaj tiv thaiv kab mob antioxidant ntawm cov protein hydrolysates thiab peptides tau tshaj tawm nyob ntawm qhov sib lawv liag ntawm cov amino acids thiab qhov loj ntawm peptide, uas cuam tshuam los ntawm hydrolysis tej yam kev mob, qhov chaw protein, thiab hom protease [35]. Raws li Adebiyi et al. [36], qhov loj tshaj plaws digestible mov bran protein tuaj yeem tawg mus rau hauv me me los ntawm subtilisin, uas ua rau muaj protein ntau thiab cov ntsiab lus. Ib qho hydrolysate's TPC thiabantioxidantKev ua haujlwm tuaj yeem cuam tshuam los ntawm qhov tshwj xeeb ntawm cov enzymes. Yog li ntawd, kev ua haujlwm antioxidant ntawm peptide tej zaum yuav cuam tshuam los ntawm cov protein cov yam ntxwv, enzyme qhov tshwj xeeb, thiab qib ntawm hydrolysis [37].
Muaj ntau cov ntawv ceeb toom siv proteases (xws li Alcalase, ib lub npe lag luam ntawm subtilisin A los ntawm hom Bacillus) kom hydrolyze cov protein los ntawm cov nroj tsuag kom tau txaisantioxidantpeptides. Nyob rau hauv no hais txog, soy proteins yog ib qho ntawm feem ntau tshaj qhia proteins [38]. Tsis tas li ntawd, Alcalase hydrolysis ntawm nplej bran protein kuj pom. Nyob rau hauv cov xwm txheej zoo, Alcalase hydrolysis ntawm glutinous nplej bran ua protein hydrolysates nrog IC50 tus nqi ntawm 0.87 士 0.02 mg/mL hauv DPPH dawb radical scavenging [39]. Hauv peb txoj kev tshawb fawb, IC50 tus nqi ntawm RPHs yog 42.58 士 2.1 mg / mL. Txawm hais tias tus nqi IC50 hauv DPPH dawb radical scavenging nyob rau hauv txoj kev tshawb no tsis zoo li uas los ntawm nplej bran protein, ABTS dawb radical scavenging (IC50=2.11 mg/mL) tau zoo dua li cov kua protein hydrolysates tau los ntawm Alcalase. hydrolysis (IC{15}}.93 mg/mL) [40].
2.3. Hyaluronidase Inhibitory Activity
Ib tug proteolytic enzyme, hyaluronidase, muaj nyob rau hauv lub dermis thiab catalyzes degradation ntawm HA nyob rau hauv lub extracellular matrix [41]. Txoj kev tshawb no siv tannic acid los ua kev tswj xyuas zoo rau kev sib piv. Daim duab 6 qhia tias tannic acid muaj ntau dua hyaluronidase kev ua haujlwm inhibition; IC50 yog 0.14 mg/mL, zoo ib yam li tus nqi tau los ntawm Nishida li al. (0.121 mg/mL; 71.1 mM) [42]. Los ntawm qhov sib piv, IC50 ntawm 8.91 mg / mL raug xam rau RPH daws. Qhov kev tshwm sim ntawm RPH cov tshuaj sib raug rau peb tus nqi IC50 dhau los, 7.61 mg / mL [43]. Proteins, polysaccharides, thiab cov nroj tsuag-originating thiab hluavtaws compounds yog nyob rau hauv ntau ntawm cov tebchaw uas muaj hyaluronidase inhibitors. Cov inhibitors pab tswj HA synthesis-degradation tshuav [44]. Tsawg HA concentration nyob rau hauv daim tawv nqaij ua rau dryness thiab wrinkles. Yog li ntawd, inhibition ntawm hyaluronidase kev ua si yog ib txoj hauv kev uas cov tawv nqaij morphology tuaj yeem txhim kho thiab nws txoj kev laus qeeb.
2.4. Tyrosinase-Inhibitory Activity
Protein hydrolysates los ntawm tej yam ntuj tso muaj peev xwm inhibittyrosinasekev ua si. Qhov kev ntsuam xyuas hauv vitro tyrosinase inhibition feem ntau yog siv los ntsuas seb cov tawv nqaij dawb li cas cuam tshuam ncaj qha rau kev ua tyrosinase [45]. Los ntawm kev koom nrog hauv melanin synthesis rate-limiting kauj ruam,tyrosinasecatalyzes L-tyrosine hydroxylation rau hauv L-DOPA thiab tom qab ntawd oxidation tom kawg rau hauv o-dopaquinone. Thaum nws tsim nyog los tiv thaiv biosynthesis ntawm melanin, inhibition ntawm L-tyrosinase kev ua haujlwm yuav yog qhov tseem ceeb. Ntawm no,tyrosinasetau siv los ua haujlwm RPH antityrosinase. Raws li tau piav qhia hauv daim duab 7, qhov concentration 107.6 mg / mL tau ua tiav 50 feem pua ntawm kev ua haujlwm ntawm tyrosinase. Ascorbic acid nthuav tawm kev ua haujlwm siab tyrosinase-inhibitory (IC50=0.098 mg/mL), uas zoo ib yam li 0.102 mg/mL uas Seo et al. qhia [46].
Daim duab 7. Cov nyhuv ntawm RPH concentration ntawmtyrosinase- inhibit kev ua haujlwm. txhais tau hais tias tsis sib koom ib tug superscript yog qhov txawv (p <>
Rice bran protein hydrolysates pom qhov tseem ceeb heevtyrosinase-inhibitory kev ua si [47,48]. Kev ua haujlwm ntawm tyrosinase-inhibitory ntawm cov tshuaj RPH tuaj yeem tshwm sim los ntawm cov amino acid profiles ntawm peptides. Schurin thiab al. piav qhia tias siv tautyrosinase-inhibitory peptides muaj arginine residues thiab phenylalanine [49]. Tyrosinase-inhibitory kev ua haujlwm tuaj yeem txhim kho los ntawm hydrophobic amino acid residues (xws li, alanine), thiab kev tsim cov melanin tuaj yeem cuam tshuam los ntawm alanine [50]. Tsis tas li ntawd, Zhang et al. kuj tau tshaj tawm tias nplej protein hydrolyzate tuaj yeem txo cov ntsiab lus melanin thiabtyrosinasekev ua haujlwm hauv UVB-induced cell qauv [51].
2.5. Amino Acid Profiles thiab MWs of RPHs
Cov nplej cov ntsiab lus protein tom qab tshem tawm cov hmoov txhuv nplej siab hauv txoj kev tshawb no yog 23.56 feem pua ntawm qhov hnyav, thiab qib ntawm hydrolysis ntawm cov qauv hydrolyzed los ntawm protease yog 9.36 feem pua. Table 1 qhia txog qhov muaj pes tsawg leeg ntawm cov amino acids hauv RPHs. Hauv cov qauv, txhua 100 g muaj 5.18 g ntawm amino acids. Hais txog cov amino acid Cheebtsam, RPHs yog nplua nuj nyob rau hauv alanine, leucine, arginine, glutamic acid, thiab aspartic acid. Txhua 100 g ntawm cov qauv muaj 1.73 g ntawm hydrophobic amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine thiab cysteine) nyob rau hauv tag nrho. Qhov tshwm sim no txawv kiag li ntawm peb daim ntawv tshaj tawm dhau los [43] hauv cov tshuaj amylase thiab nws qhov kev kho kub kom tshem tawm cov hmoov txhuv nplej siab. Cov ntsiab lus ntawm hydrophobic amino acids yog 1.90 npaug ntau dua li peb daim ntawv tshaj tawm dhau los. Qhov ntsuas kub qis dua (60 ℃) hauv txoj kev tshawb no tuaj yeem tiv thaiv denaturation ntawm cov protein ntau, kom cov haujlwm ntawm cov amino acids yuav khaws cia zoo dua. Tsis tas li ntawd, cov lus xaus zoo sib xws kuj tau txais los ntawm lwm cov kab mob fungal amylase thiab glucoamylase rau saccharify starch hauv cov nplej dawb [52].
Table 1. Amino acid cov qauv ntawm nplej protein hydrolyzate (RPH) cov qauv.
Kev tshawb fawb tau pom hydrophobic amino acids zoo liantioxidantslos ntawm kev nce lipid-raws li solubility hauv cov protein hydrolysates thiab peptides los ntawm ntau qhov chaw protein, yog li txhawb kev sib cuam tshuam nrog cov dawb radicals [38,53]. Qee cov amino acids tau tshaj tawm los ntawm Chen li al. [54] feem ntau yog antioxidants; Cov kua qaub uas tau hais muaj xws li tryptophan, cysteine, methionine, tyrosine, thiab histidine. Hauv txoj kev tshawb no, cov amino acids uas muaj ntxhiab (phenylalanine, tyrosine, thiab tryptophan) muaj 0.53 g / 100 g RPHs. Yog li ntawd, cov peptide-originating amino acids yog tej zaum lub luag hauj lwm rau RPHs 'antioxidantkev ua si.
Tsis tas li ntawd, cov proteins uas yog hydrolyzed rau hauv luv luv peptides muaj qhov sib txawv MW faib, thiab qee pawg hydrophobic folded nyob rau hauv sab hauv ntawm tag nrho cov natural protein molecules feem ntau raug rau cov theem aqueous. Qhov no muaj feem xyuam rau cov protein molecules uas raug structurally rearranged thiab yog li ntawd rau cov protein cov functional properties [55,56]. Cov ntaub ntawv tricine-SDS-PAGE qhia tias MW ntawm RPHs nyob hauv thaj tsam 5–35 kDa (Daim duab 8a).
Daim duab 8. Kev txiav txim siab Molecular hnyav (MW) rau RPHs los ntawm sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (SDS-PAGE): (a) tricine-SDS-PAGE cov qauv ntawm MW marker (kab 1) thiab RPHs (kab 2) thiab (b) cov ntsiab lus txheeb ze (%) ntawm txhua MW ntawm RPHs.
Daim duab 8b qhia txog cov ntsiab lus txheeb ze ntawm ntau yam MWs hauv RPHs. Zuag qhia tag nrho, 45.24 feem pua ntawm tag nrho cov protein nyob hauv lub ntsiab band (MW ≈ 2.4 kDa). Cov txiaj ntsig zoo sib xws tau txais txog cov nplej bran protein hydrolysates 'peptide. Qhov siab tshajantioxidantkev ua tau los ntawm Thamnarathip et al. [37] yog rau peptides ntawm MW=6–50 kDa. Tsis tas li ntawd, kev sib raug zoo ntawm cov protein hydrolysates 'kev ua haujlwm thiab MW faib thiab muaj pes tsawg leeg ntawm cov amino acids [57]. Qhov no piav txog qhovantioxidantKev ua haujlwm ntawm RPHs pom nyob rau hauv txoj kev tshawb no.
2.6. Cell Toxicity Test
Tsawg cell toxicity yuav tsum tau siv rau yav tom ntej. Txhawm rau ntsuas cytotoxicity thiab biocompatibility ntawm RPHs, cell viability ntawm raw 264.7 hlwb hauv RPH tov tau ntsuas los ntawm MTT txoj kev. Raws li pom nyob rau hauv daim duab 9, lub cell viability siab tshaj 100 feem pua thaum kho nrog 25–2000 µg/mL RPH rau 24 h thiab 48 teev. Cov txiaj ntsig qhia tau hais tias tsis tshua muaj cytotoxicity ntawm RPHs. Yog li, RPHs tuaj yeem siv tau los ua cov ntawv thov kom zoo nkauj nrog cytotoxicity tsawg heev.
Daim duab 9. MTT kev soj ntsuam rau cov cell toxicity ntawm raw 264.7 hlwb kho nrog sib txawv RPH concentrations rau 24 thiab 48 teev.
3. Cov ntaub ntawv thiab cov txheej txheem
3.1. Reagents
Iron (III) chloride, 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Trolox (6-hydroxy-2,5,7 , 8-tetramethylchroman-2-carboxylic acid), l-3, 4-dihydroxyphenylalanine (L-DOPA), 1,1-diphenyl-2-picrylhydrazyl ( DPPH), thiab trichloroacetic acid tau txais los ntawm Alfa Aesar (Tewksbury, MA, USA). 2,2′ -azobis(2-methylpropionamidine) dihydrochlo- caij (AAPH), Folin-Ciocalteu's phenol reagent, gallic acid, ascorbic acid, nceb ty-rosinase, thiab uorescein sodium tau txais los ntawm Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate tau txais los ntawm Riedel-de Haën (Seelze, Lub Tebchaws Yelemees). Thaum kawg, potas-sium ferricyanide, sodium hydrogen phosphate, thiab sodium dihydrogen phosphate tau txais los ntawm Showa Chemical (Tokyo, Nyiv).
3.2. Kev npaj ntawm RPHs
RPHs tau npaj raws li tau piav qhia yav dhau los, tshwj tsis yog tias cov kab mob fungal amylase tau raug coj los ua kom cov hmoov txhuv nplej siab hauv cov nplej nplej, zam kev puas tsuaj rau cov amino acids los ntawm cov kab mob amylase hydrolysis ntawm qhov kub thiab txias [43,58]. Ib puas grams nplej tau tsau rau hauv 1000 mL ntawm cov dej distilled uas muaj 0.5 feem pua ntawm cov kab mob fungal amylase (Genencor, NY, USA); Qhov sib tov tau muab tso rau hauv qhov kub ntawm 24 teev txog 60 ℃ (pH 4.2), tom qab ntawd nws raug tso cai kom txias rau chav sov. Centrifugation tau ua rau 10 min ntawm 1968 × g kom tshem tawm cov supernatant ntxiv. Tom qab 20- quav dej thiab 2 mL ntawm 0.1 feem pua ntawm hydrolytic protease (Healthmate, Changhua, Taiwan) tau ntxiv rau qhov tsis sib haum, cov tshuaj tau shaken thiab incubated rau 4 h ntawm 55 OC C. Txoj kev pH-stat yog ua hauj lwm los tswj cov kev daws teeb meem ntawm pH ntawm qhov zoo tshaj plaws, thiab 85 О C cua sov ces ua rau 10 min rau enzyme inactivation. Qhov seem insoluble feem raug tshem tawm los ntawm centrifugation rau 15 min ntawm 3075 × g. Lyophilization tau ua tiav ntawm lub supernatant, uas tau muab khaws cia ntawm _20 О C ua ntej siv.
3.3. Antioxidant Kev Ua Haujlwm ntawm RPHs
3.3.1. Tag Nrho Phenolic Concentration (TPC)
Folin-Ciocalteu txoj kev tshawb nrhiav TPC ntawm RPHs tau ua haujlwm [59]. Ua ntej, 200 µL ntawm Folin-Ciocalteu's phenol reagent (0.3M) tau sib xyaw ua ke los ntawm 5-min co nrog 200 µL ntawm RPH kua, thiab rau qhov sib tov no, 400 µL ntawm deionized (DI) dej thiab 200 µL ntawm 10 feem pua (w/v) sodium carbonate tov tau ntxiv. Cov tshuaj tov tau underwed 60 min ntawm incubation nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub. Tom qab ntawd nws tau centrifuged rau 15 min ntawm 3000 rpm. Kev ntsuas tau siv 100 µL ntawm supernatant. Txhawm rau txiav txim siab TPC (unit: mg) ntawm qhov sib npaug ntawm gallic acid (GAE) ib gram ntawm cov qauv RPH qhuav (unit: mg GAE / g RPHs), cov ntaub ntawv kho qhov muag tau nkag mus rau tus qauv nkhaus sawv cev rau gallic acid. Lub absorbance tau txais ntawm 700 nm los ntawm kev siv Epoch Microplate Spectrophotometer (BioTek, VT, USA).
3.3.2. Tag nrho cov ntsiab lus Flavonoid (TFC)
TFC tau txais tom qab txoj hauv kev ntawm Wathoni li al. nrog me me kev hloov kho [60]. Ua ntej, 500 µL txhua tus qauv thiab 2 feem pua (w / v) aluminium chloride tov tau tov. Cov tshuaj tiv thaiv tau muab sib xyaw kom huv si thiab sab laug rau 10 feeb, thiab qhov nqus ntawm 415 nm raug soj ntsuam. Cov txiaj ntsig tau tshaj tawm hauv micrograms ntawm quercetin sib npaug (QE) ib gram ntawm cov qauv RPH qhuav (µg QE / g RPHs).
3.3.3. DPPH Dawb Radical Scavenging Activity
Ua ntej, 198 µM DPPH ethanol tov (50 µL) thiab RPH tov los yog DI dej (0.5 µL; cov qauv thiab tswj, raws li) tau sib tov thiab ces cia sawv ntsug rau 30 min nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub. Tom qab ntawd cov tshuaj absorbance ntawm 517 nm tau txais. Cov txheeb ze scavenging kev ua si tau suav los ntawm kev txiav txim siab qhov sib txawv ntawm qhov nqus ntawm cov qauv thiab kev tswj. High DPPH dawb radical scavenging kev ua ub no tau cuam tshuam los ntawm qhov muag tsis pom kev. Hauv kev tshuaj xyuas RPH's DPPH dawb radical scavenging kev soj ntsuam, tus qauv ua haujlwm yog vitamin C [61–63].
3.3.4. Kev Tshawb Fawb Kev Ua Haujlwm ntawm ABTS Dawb Radicals
Txoj hauv kev qhia los ntawm Wu et al. tau ua hauj lwm los ntsuam xyuas cov kev daws teeb meem RPHantioxidantkev ua si [64]. Ua ntej, 7 mM ABTS stock solution (250 µL) tau tshwm sim nrog 2.45 mM potassium persulfate (250 µL) kom tawm los ntawm ABTS dawb radical cation (ABTS● ntxiv), nrog rau cov sib tov khaws cia. rau 16 teev ntawm 4 ℃ hauv qhov tsaus ntuj ua ntej nws ua haujlwm. Tom qab sib npaug hauv qhov tsaus ntuj ntawm chav tsev kub, 0.1 M phosphate-buffered saline (PBS; pH 7.4) tau ua haujlwm kom dilute cov tshuaj rau 0.70 士 0.02 absorbance ntawm 734 nm. Tom qab ntawd, rau 180 µL ntawm diluted ABTS tov, 20 µL ntawm Trolox (zoo tswj) lossis RPH tov (piv txwv) tau ntxiv. Tom qab ntawd qhov sib tov tau raug rau 10 min ntawm chav tsev kub incubation. Txoj kev tshawb no tau txiav txim siab qhov muag pom ntawm 734 nm; qis absorbance sib raug rau siab dua ABTS dawb radical scavenging kev ua. Tus qauv siv rau kev ntsuam xyuas RPH kev daws teeb meem ABTS dawb radical scavenging kev ua si yog cov antioxidant Trolox.
3.3.5. Txo peev xwm
Kev ntsuam xyuas ferric-txo antioxidant zog tau ua haujlwm los txiav txim siab RPH cov tshuaj tag nrhoantioxidantkev ua si. Raws li qhia los ntawm Lin et al. [29], RPH tov (200 µL) yog sib xyaw nrog 1 feem pua (w/v) K3Fe(CN)6 thiab 0.2 M PBS tsis (pH 6.6; 100 µL txhua) . Rau 20 min, ib lub da dej 50 OC tau ua haujlwm los ua kom sov qhov sib tov; Tom qab tshem tawm cov sib tov los ntawm da dej, nws tau txias sai sai rau 3 min. Tom qab ntawd, ntxiv 10 feem pua (w/v) trichloroacetic acid (100 µL) thiab 10-min centrifugation ntawm 3000 rpm tau ua. Qhov no tau ua raws li kev rho tawm ntawm supernatant (400 µL) thiab nws cov khoom sib xyaw nrog 0.1 feem pua (w/v) FeCl3 (100 µL) thiab DI dej (400 µL). Fe4 [Fe(CN)6]3 tau los ntawm 10-min cov tshuaj tiv thaiv ntawm qhov sib tov hauv qhov tsaus ntuj. Tom qab ntawd, qhov siab dua qhov muag pom ( ntsuas ntawm 700 nm) qhia tias muaj peev xwm txo qis dua. Tus qauv vitamin C tau siv los txiav txim siab cov vitamin C sib npaug (VCE) cov ntsiab lus ib gram ntawm RPHs.
3.3.6. Oxygen Radical Absorbance Peev Xwm (ORAC)
Txoj kev tshawb no tau txais ORAC los ntawm kev hloov kho ib txoj kev qhia yav dhau los [65]. Tom qab sib tov ntawm cov qauv RPH hauv cov dej distilled, RPH tov (50 µL) tau tov nrog fluorescein (10 µM) hauv 96-zoo microtiter phaj. Cov kev daws teeb meem tau ua tiav 15-min incubation ntawm 37 OC C ua raws li qhov sib ntxiv ntawm 50 µL ntawm AAPH (500 mM). Txhua 5 min thiab tshaj li ntawm 120 min, cov fluorescence tau sau tseg (λex thiab λem=485 thiab 528 nm, feem). CovantioxidantLub peev xwm ntawm RPHs tau tshawb pom los ntawm fluorescence decay kinetics los ntawm kev suav thaj tsam hauv qab nkhaus (AUC). Hauv kev suav RPH ORAC, tus qauv yog 15-250 µM Trolox. ORAC tau tshaj tawm tias yog micromoles ntawm Trolox sib npaug (TE) ib gram ntawm qhuav RPH qauv (µmol TE / g RPHs).
3.4. Hyaluronidase Inhibitory Activity
Kev kuaj hyaluronidase inhibition tau ua tiav siv {{0}}zoo microplate thiab ib txoj kev qhia yav dhau los nrog kev hloov kho me ntsis [40]. N-acetylglucosamine tau tso tawm los ntawm reacting hyaluronidase nrog HA substrate. Nyob rau hauv lub xub ntiag ntawm ib qho inhibitor, N-acetylglucosamine tso tawm raug txo, nrog rau qhov kev tso tawm no los ntawm kev tau txais cov 600-nm absorbance. HA tau precipitated nrog acidic albumin tov muaj li ntawm 0.1 M acetate tsis (pH 3.9) thiab bovine serum albumin (1 mg/mL). Cov qauv tshuaj thiab 5 mg/mL hyaluronidase underwent 20-min incubation ntawm 37 OC C. Rau incubation sib tov, HA (1{{20}}}0 µL; 5.0 mg/mL hauv 0.1 M acetate buffer) tau ntxiv tom qab. Ntxiv incubation ntawm 37 ℃ rau 40 min tau ua. 0.1 mL 0.4 M alkaline borate tov tau ntxiv los nres cov tshuaj tiv thaiv enzymatic.
3.5. Tyrosinase-Inhibitory Activity
Qhov kev tshawb fawb tam sim no tau soj ntsuam covantityrosinasekev ua ntawm RPHs los ntawm kev siv cov txheej txheem tshaj tawm yav dhau los nrog kev hloov kho [66]. Ib qho tshuaj enzyme (135 U / mL) tau npaj los ntawm kev sib caistyrosinasenyob rau hauv 20 mM phosphate tsis (pH 6.8). Tsis tas li ntawd, DI dej tau ua hauj lwm rau 1.25 mM L-DOPA tov npaj. Tom qab ntawd, 40 µL ntawm ntau qhov ntau ntawm RPH cov qauv kev daws teeb meem tau sib xyaw nrog 40 µL ntawm cov tshuaj tyrosinase thiab 120 µL ntawm L-DOPA daws. Rau 30 feeb, qhov sib tov no tau khaws cia ntawm 37 ℃ hauv kev sim ntawm RPHs ' inhibition ntawmtyrosinasekev ua si. Lub spectrophotometer (FLUOstar Omega Microplate Reader, BMG Labtech GmbH, Lub teb chaws Yelemees) tau ua haujlwm kom tau txais 475-nm absorbance. Tag nrho cov kev ntsuas tau ua peb zaug. Lub absorbance ntawm cov coj pawg thaumtyrosinasetsis yog tam sim no raug rho tawm. Lub enzyme inhibition tus nqi tau txiav txim siab li
3.6. Characterization ntawm RPHs
3.6.1. Amino Acid Profiles
Txoj kev tshawb no tshawb pom cov amino acid muaj pes tsawg leeg ntawm RPHs. Ua ntej, rau 24 teev thiab ntawm 115 ℃, 4 M methanesulfonic acid tau ua haujlwm rau hydrolyze cov qauv hauv evacuated kaw hlab. Ob Lub Dej 510 hnyav xa cov tshuab thiab lub tshuab ntsuas amino acid (L- 8900; Hitachi, Tokyo, Nyiv) tau ua haujlwm rau kev sib cais cov amino acid sib cais ntawm Spherisorb ODS2 kem ntsuas 25 m × 64.6 mm. Txoj kev tshawb no siv cov kuab tshuaj hauv qab no: (a) sodium acetate (0.14 M) thiab triethylamine (850 µL/L; pH 5.6) thiab (b) 60 feem pua acetonitrile, uas cov gradient yog 0 feem pua rau 2 min; 0-42 feem pua rau 15.5 min (convex nkhaus); thiab 100 feem pua rau 4 min. Cov qauv piv txwv tau muab coj los ntsuas rau kev ntsuas ntawm cov amino acid profile ntawm 254 nm [67,68].
3.6.2. Molecular Luj (MW) ntawm Protein
Raws li Schägger txoj kev [69] thiab nyob rau hauv kev txo qis, txoj kev tshawb no tau txais MW faib los ntawm tricine-sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) nrog kev hloov kho me ntsis. Ib qho piv txwv tsis muaj (30} g/L SDS, 0.375 M Tris-HCl, 0.125 g/L Coomassie Brilliant Blue G-250, thiab 75 g /L glycerol; pH 7) tau ua haujlwm kom tawg cov qauv khov-qhuav, nrog centrifugation ces ua ntej thauj khoom. Tag nrho ntawm 20 µL 2-mercaptoethanol tau ntxiv rau 1 mL ntawm cov qauv tricine-SDS-PAGE. Cov qauv yog rhuab ntawm 100 ℃ rau 90 s. Ib qho piv txwv zoo tau thauj khoom nrog txhua tus qauv thiab Cov Khoom Siv Tsis Muaj Cov Khoom Uas Muaj Cov Txheej Txheem Dav Dav (Bio-Rad Laboratories, Lub Tebchaws Yelemees) los ntawm kev siv lub microsyringe. Electrophoresis tau ua tiav - thawj zaug ntawm qhov tsis tu ncua 30 mV kom txog rau thaum tag nrho cov qauv muaj nyob rau hauv stacking gel thiab tom qab ntawd kom txog rau thaum ua tiav ntawm 100 mV tas li. Tom qab ntawd, 0.02 feem pua Coomassie Brilliant Blue R-250 daws tau siv rau gel staining. Cov keeb kwm yav dhau destaining ntawm cov gels tau ua los ntawm co cov gels hauv 10 feem pua acetic acid thaum hmo ntuj. Thaum kawg, cov duab gel tau txheeb xyuas los txheeb xyuas cov kab protein nyob hauv txoj kab; Qhov kev tshuaj ntsuam no tau ua nyob rau hauv ImageJ (US National Institutes of Health, Bethesda, MD, USA). Cov cim cim tau ua haujlwm kom tau txais ib qho kev ntsuas qhov nkhaus uas qhov MW tau kwv yees. Luv luv, thawj kauj ruam yog los txiav txim siab txhua pab pawg qhov ntev ntawm kev tsiv teb tsaws (Rf) los ntawm sab saum toj ntawm qhov sib cais gel. Cov kauj ruam thib ob yog kev suav ntawm qhov ntsuas qhov nkhaus los ntawm kev siv Rf thiab log (MW) rau tus qauv cim nrog ib qho MW. Kev txiav txim siab MW tau ua tiav siv Rf ntawm cov protein ntau hauv RPHs.
3.7. Cytotoxicity Assay
Raw 264.7 hlwb raug coj mus kuaj hauv cov piam thaj siab Dulbecco's Modified Eagle Medium (DMEM) uas muaj 10 feem pua fetal bovine serum (FBS), 4.5 g/L Glucose, 1 feem pua cov tshuaj tua kab mob (100 units/ mL penicillin thiab 100 µg / mL Streptomycin), 4 mM L-Glutamin thiab 1.5 g / L sodium bicarbonate ntawm 37 OC thiab 5% CO2. Lub cell toxicity ntawm raw 264.7 hlwb rau RPHs yog ntsuas los ntawm ib tug 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) proliferation assay txoj kev. Kwv yees li 1 × 104 hlwb ntawm ib qhov dej tau plated nyob rau hauv 96-zoo daim hlau. Tom qab 24 teev, ntau qhov ntau ntawm RPHs (0-2000 μg / mL) tau ntxiv rau hauv cov hlwb. Tom qab 24 thiab 48 teev ntawm incubation, 100 µL ntawm MTT tov (0.5 mg / mL) tau ntxiv. Blue formazan crystals tau pom thaum kuaj xyuas hauv lub tshuab tsom. DMEM raug tshem tawm thiab 100 µL ntawm dimethyl sulfoxide (DMSO) tau ntxiv rau ib qhov dej. Qhov nqus tau raug ntsuas los ntawm kev nyeem ntawv microtiter phaj. Lub cell viability (feem pua ) yog ces xam raws li [A570 (kho cov hlwb) _ A570 (tom qab)] / [A570 (tsis kho hlwb) _ A570 (tom qab)] × 100 feem pua [70].
3.8. Kev txheeb cais
Daim ntawv tshaj tawm rau txhua tus qauv hydrolyzate yog tus nqi nruab nrab los ntawm peb txoj kev ywj pheej rov ua dua thiab kev txiav txim siab. Cov txiaj ntsig tau hais tawm hauv qhov txhais tau tias 士 tus qauv sib txawv (SD) tau txheeb xyuas los ntawm ib-txoj kev ANOVA thiab Duncan's post hoc xeem uas siv cov Kev Tshawb Fawb Txog Kev Tshawb Fawb (version 20.0; SPSS, Armonk, NY, USA) . Tus nqi ntawm p <0.05 tau="" suav="" tias="" yog="" qhov="" tseem="" ceeb="" ntawm="" kev="" txheeb="">
4. Cov lus xaus
Txoj kev tshawb no tau tshuaj xyuas cov haujlwm ntawm RPHs. Cov txiaj ntsig kev sim tau qhia tias RPHs muaj cov tshuaj phenolic thiab flavonoids thiab nthuav tawm ntau yamantioxidantkev ua ub no, xws li DPPH thiab ABTS scavenging kev ua ub no, txo peev xwm, thiab ORAC. Tsis tas li ntawd, RPHs zoo inhibitedtyrosinasethiab hyaluronidase kev ua ub no. Cov protease yog qhov tseem ceeb cuam tshuam rau MW qauv ntawm RPHs. Kev tsom xam ntawm RPHs qhia lawv lub peev xwm siv los ua cov khoom hauv cov tshuaj pleev ib ce.
Sau Kev Koom Tes: Txoj kev tshawb no tau tsim los ntawm Y.-SL; kev soj ntsuam raug ua los ntawm Y.-SL thiab C.-SC; cov ntaub ntawv curation tau ua los ntawm H.-JC, F.-JD, C.-YC, J.-HZ thiab Y.-CH; cov ntawv sau qub yog tsim los ntawm H.-JC, F.-JD, thiab S.-LF; Thaum kawg, tsab ntawv tau tshuaj xyuas thiab kho los ntawm C.-FC, Y.-SL, thiab C.-SC Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.
Kev Kawm Txuj Ci: Ministry of Science thiab Technology tau muab nyiaj pab rau txoj kev kawm no. Institutional Review Board Statement: Tsis siv tau.
Cov Lus Qhia Txog Kev Pom Zoo: Tsis siv tau.
Daim Ntawv Qhia Txog Cov Ntaub Ntawv: Cov ntaub ntawv qhia hauv qhov kev tshawb fawb no muaj nyob rau ntawm kev thov los ntawm tus neeg sau ntawv.
Kev tsis sib haum xeeb ntawm kev txaus siab: Cov kws sau ntawv tshaj tawm tias tsis muaj kev cuam tshuam ntawm kev txaus siab. Qauv Muaj: Tsis muaj.
