Antioxidant Thiab Anti-aging Potential Ntawm Peptide Formulation (Gal2-Pep) Conjugated Nrog Gallic Acid Ⅱ
May 04, 2023
3. Cov txiaj ntsig thiab kev sib tham
3.1. Synthesis thiab conjugation ntawm gallic acid nrog peptide
Cov synthesized peptides TPPTTP, galloyl-TPPTTP (Gal-Pep), thiab Gal2-Pep tau txais thiab purified siv semi-preparative RP-HPLC. Fig. 1a thiab Scheme 1 qhia cov tswv yim sib txuas ntawm gallic acid nrog KTPPTTP li Gal2-Pep. Hauv SPPS txoj kev, cov impurities tsim thaum lub sij hawm peptide synthesis feem ntau muaj xws li sab-reacted peptides, amino acid-tiv thaiv pawg, thiab cov kuab tshuaj siv rau synthesis thiab purification. Lub HOBt siv hauv peptide synthesis paub tias inhibit racemization thiab txhim kho kev ua haujlwm ntawm peptide synthesis.17-19 Txhawm rau tshem tawm cov amino acid tiv thaiv pawg thiab cov kuab tshuaj siv rau kev sib txuas, peb tau ntxuav ob peb zaug siv cov kuab tshuaj xws li DMF thiab DCM thaum lub sijhawm cov txheej txheem peptide synthesis. Txawm tias tom qab qhov kawg ntxuav cov kauj ruam, impurities nrog rau cov amino acid-tiv thaiv pab pawg tseem muaj nyob rau hauv kab. Txhawm rau kom ua tau raws li peptide purity ntawm ntau dua 90 feem pua tau tshaj tawm los ntawm ntau tus kws tshawb fawb, 20-22 semi-prep HPLC tau ua raws li cov kauj ruam polishing kom tau txais nal peptide ntawm 99.2 feem pua purity raws li kev txheeb xyuas HPLC (saib daim duab. S1†). Cov khoom purified peptide tau soj ntsuam siv Q-TOF kom paub tseeb tias lawv cov kev ua tiav tiav (Fig. 1b–d).
Nyem qhov no kom tau txais cov ntaub ntawv ntau ntxiv txog Cistanche Treat Anti-Aging
3.2. Cell viability assay
3.3. Antioxidant kev ua ntawm synthesized peptides
TPPTTP tsis muaj qhov cuam tshuam loj heev ntawm kev ua kom pom tseeb, thaum GA, Gal-Pep, thiab Gal2-Pep tau nthuav tawm ntau dua radical scavenging kev ua si nyob rau hauv ib tug concentration-dependent yam.
Hauvtshwj xeeb, Gal2–Pep tau pom qhov ua tau zoo tshaj plawsantioxidantkev ua si; nyob rau hauv ib tug concentration ntawm 10mM, GA, Gal–Pep, thiab Gal2–Pepmuaj ib qho kev tawm dag zog ntawm 13.6 feem pua, 24.23 feem pua, thiab 26.0 feem pua ,raws. Qhov no qhia tau hais tias kev khi ntawm TPPTTP rau GA uatsis inhibit radical scavenging kev ua. Fig. 3b confi uarms uastability ntawm cov qauv raws li confi uarmed los ntawm kev ntsuam xyuas lawv cov radicalscavenging kev ua si thaum khaws cia hauv chav tsev kub. GA khaws cianws radical scavenging kev ua si mus txog rau ib lub lim tiam tab sis, aftyog ob lub lis piam, qhov haujlwm no tau txo qis dua 60 feem pua. Ntawm lwm yamtes, Gal–Pep thiab Gal2–Pep tuav rawv lawv txoj haujlwm khawb avrau ntau tshaj ob lub lis piam. Hauv particular, Gal2–Pep muaj radical scavenging kev ua ntawm 50 feem pua txawm tias nyob rau hauv nws lub lim tiam plaub. Cov txiaj ntsig noqhia tias kev khi GA rau TPPTTP ruaj khov nws qhov kev tshem tawm radicalkev ua si. Qee qhov kev tshawb fawb tau pom tias ascorbic acid ruaj khovlos ntawm kev khi nws nrog peptides.12,25 Peb ntseeg tias GA yuav nyob ruaj khovlized los ntawm khi nws nrog ib tug peptide nyob rau hauv tib txoj kev.
Covantioxidant nyhuv ntawm peptideskuj tau sim siv DCF DA kev soj ntsuam nyob rau hauv lub xub ntiag ntawm intracellular oxidative kev nyuaj siab tshwm sim los ntawm H2O2 (Fig. 4). Thaum 24 teev ntawm kev kho cov qauv ntawm cov hlwb ntawm qhov concentration ntawm 100 mM, DCF-DA tau thauj khoom mus rau lub hlwb, thiab cov hlwb raug kho nrog H2O2 rau 30 min thiab qhov kev siv fluorescence ntawm DCF tau pom ntawm qhov ua kom zoo siab. wavelength ntawm 485nm thiab emission wavelength ntawm 535nm. Cells raug oxidative kev nyuaj siab nrog 1 mM H2O2 nthuav tawm fluorescence siv uas ntau tshaj peb zaug siab tshaj qhov tsis zoo tswj; Txawm li cas los xij, kev kho Gal2-Pep tau tsim cov fluorescence uas tsuas yog 67 feem pua ntawm cov uas tsim los ntawm cov hlwb kho nrog H2O2 nkaus xwb. Nyob rau hauv lub xub ntiag ntawm TPPTTP, uas tsis tshwm sim radical scavenging kev ua si (Fig. 3a), ROS kev nyuaj siab tshwm sim los ntawm H2O2 raug txo los ntawm kwv yees li 20 feem pua, txhawb kev tshawb fawb yav dhau los uas tau qhia tias TPPTTP txo ROS theem hauv hlwb.24 Thaum lub hlwb raug kho nrog GA nkaus xwb, tsis muaj qhov hloov pauv tseem ceeb hauv qib ROS. Ua ke, cov txiaj ntsig no qhia tau tias Gal2-Pep, uas ua ke ob lub GA molecules nrog peptide, yog ib qho kev ruaj khov thiab zoo antioxidant.
3.4. Cov nyhuv ntawm synthesized peptides ntawm mitochondria membrane muaj peev xwm
Hauv txoj kev tshawb no, JC-1 dye tau siv los tshawb xyuas cov txiaj ntsig ntawm cov peptides synthesized ntawm MmP (DJm). MmP ua raws li qhov tseem ceeb tsis zoo rau kev ua haujlwm mitochondrial thiab paub tias muaj feem xyuam nrog ntau yam kab mob xws li Alzheimer's thiab Huntington's disease.26 JC-1 dye ua ib lub tsho liab los ntawm cov emissions ntsuab los ntawm accumulating nyob rau hauv mitochondria thiab tsim J-aggregates. Tom qab kho cov HaCaT thiab fibroblast hlwb nrog cov synthesized peptides rau 24 h, cov hlwb raug rau JC-1 zas rau 10 min, thiab tom qab ntawd cov fluorescence siv tau ntsuas siv lub microplate nyeem ntawv (ntsuab lEx ¼ 475 nm thiab lEm. ¼ 530 nm, liab lEx ¼ 475 nm thiab lEm ¼ 590 nm). Qhov piv ntawm cov xim liab rau ntsuab fluorescence tau qhia raws li qhov hloov pauv piv rau kev tswj tsis zoo (Daim duab 5). HaCaT hlwb kho nrog GA thiab peptide ntawm nws tus kheej tsis pom qhov hloov pauv tseem ceeb piv rau pawg tswj tsis zoo, thaum Gal2-Pep nthuav tawm qhov sib txawv tseem ceeb, nce 1.45-fold piv rau kev tswj. Cov hlwb fibroblast kho nrog Gal2-Pep kuj tseem ceeb heev
Daim duab 4 Intracellular antioxidant kev soj ntsuam siv DCF-DA ntawm gallic acid thiab peptide concentrations ntawm 100 mM. Tom qab 24 teev ntawm kev kho mob, H2O2 tau ntxiv thiab incubated rau 30 feeb, ua raws li kev ntsuas ntawm DCF fluorescence siv. Asterisks qhia txog qhov sib txawv tseem ceeb (*p <0.05, **p <0.005, ***p <0.001).
3.5. Elastase inhibitory kev ua haujlwm
3.6. Kev nthuav qhia ntawm hom I collagen, MMP-1, thiab PGC-1ib noob
Fig. 7 Gene expression analysis in dermal fibroblasts using RT-qPCR: (a) type I collagen, (b) MMP-1, and (c) PGC-1a.
Qhov kev qhia ntawm PGC-1a tau siab tshaj tom qab 8 teev rau txhua qhov kev kho mob, nrog rau kev qhia tus nqi rau GA, TPPTTP, Gal–Pep, thiab Gal2–Pep nce 2.06-, 1.{{6} }, 1.07-, thiab 2.05-fold, ntsig txog. Txawm li cas los xij, tsis zoo li lwm yam kev kho mob, Gal2-Pep nce PGC-1 ib qho kev qhia 1.27-fold tom qab 24 teev. PGC-1atswj cov synthesis thiab antioxidant teebmeem ntawm mitochondriathiab tau tshaj tawm rautxo nrog kev laus.29–32 Yog li ntawd, Gal2–Pep muaj peev xwm siv tau los ua cov tshuaj antioxidant uas tuaj yeem tiv thaiv ROS zoo.
Muab tag nrho ua ke, Gal2-Pep tau nce qhov kev qhia ntawm PGC-1a thiab collagen piv rau cov teebmeem ntawm GA thiab TPPTTP ib leeg, thaum lubkev qhia ntawm MMP-1 zoo li txo qis, yog li qhia txog nws lub peev xwm los siv hauvanti-aging tshuaj pleev ib ce.
4. Cov lus xaus
Kev tsis sib haum xeeb
Tsis muaj kev tsis sib haum xeeb los tshaj tawm.
Kev lees paub
Txoj kev tshawb no tau txais kev txhawb nqa los ntawm qhov nyiaj pab ntawm Kauslim Industrial Complex Corp. (KICOX, NTIS No. 1415165722) thiab Inha University Research Grant.
Cov ntaub ntawv
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23 DJ Tobin, J. Tissue Viability, 2017, 26, 37–46.
