Antioxidant Thiab Anti-aging Muaj peev xwm ntawm Peptide Formulation (Gal2-Pep) Conjugated Nrog Gallic Acid

Daim tawv nqaij yog qhov yooj yim heev rau kev laus ntxov ntxovvim yog kev ntxhov siab sab nraud, yog li ntawd, hauv txoj kev tshawb no, peptide formulation, (galloyl)2–KTPTP (Gal2–Pep) tau tsim los ntawm kev sib txuas TPPTTP peptide, thiab gallicacid (GA). Tag nrho cov peptides tau tsim los ntawm 2-chlorotrityl chloride resin siv cov khoom-theem peptidesynthesis (SPPS), thiab soj ntsuam ntawm electrospray ionization (ESI) / quadrupole-time-of-flfl uaight (Q-TOF) tandem loj spectroscopy (MS) system. Thaum pib, Gal2–Pep pom tsis muaj toxicity hauv qab qhov concentration 100mM nrog tus nqi ntawm cov ciaj sia taus ntawm 88 feem pua ​​​​rau keratinocytes thiabfibroblasts. Covreactive oxygen hom(ROS) kev ua si ntawm Gal2–Pep tau ruaj khov dua piv rau GA ib leeg; thiab tom qab plaub lub lis piam hauv chavkub, nwsROS scavenging kev ua sitseem siab dua 50 feem pua. Ntxiv mus, peptide formulation,Gal2–Pep kuj tau nthuav tawm cov nyhuv elastase inhibitory hauv CCD-1064Skfibroblasthlwb. Raws li cov txiaj ntsig ntawm RT-qPCR, nws tau ua pov thawj hauv qhov kev tshawb fawb no tias Gal2–Pep nce qhov kev qhia ntawm PGC-1a rautiv thaivoxidative kev nyuaj siab, thiab validated nws lub peev xwm raws li ib tuganti-aging agentlos ntawmnce kev nthuav qhia ntawmtype I collagenthiab los ntawmtxo qhov kev qhia ntawm matrix metalloproteinase-1 (MMP1). Covkev tshawb pom tau txais kev txhawb nqa cov lus qhia tias peptide formulation synthesized nyob rau hauv txoj kev tshawb no yuav siv tau raws li ib tugnatural antioxidantthiabanti-aging agentrau nws cov kev siv tshuaj pleev ib ce.

Nyem qhov no Kom Tau Txais Cov Lus Qhia Ntxiv Txog Cistanche Anti-Aging Products

1. Taw qhia

Cov tawv nqaij laustshwm sim los ntawm kev maj mam txo qis thiab qhov kawg nres ntawm kev faib cell ntawm keratinocytes thiab bro.blasts nyob rau hauv daim tawv nqaij. Wrinkles ntawm daim tawv nqaij loj hlob raws li tus naj npawb ntawm cov hlwb txo, nrog rau qhov tshwm sim denaturation ntawm extracellular matrix ua rau dryness thiab poob ntawm elas.ticity nyob rau hauv daim tawv nqaij.1 Kev puas tsuaj rau intracellular mitochondrial DNAthiab kev txo qis ntawm cov protein synthesis kuj tshwm simthaum lub sij hawm daim tawv nqaij laus txheej txheem.2 Kev laus ntawm daim tawv nqaij muaj ob txoj hauv kevtxoj kev, intrinsic thiab extrinsic, nrog ultraviolet (UV) raug

tus tsav tsheb loj ntawmextrinsic laus. Lub teeb UV reacts nrog cov khoom tseem ceeb ntawm daim tawv nqaij, suav nrog lipids, proteins, thiab nucleic acids, los tsim cov pa oxygen reactive (ROS) uas ua rau cov cell.tuag.3–5 Ntau ROS ntau ntau kuj ua rau cov cleavage thiabQhov txawv txav ntawm kev khi ntawm collagen los yog elastin chains thiab txhawb kev qhia ntawm matrix metalloproteinases (MMPs), xws li MMP-1, uas ua rau collagen, ua rau wrinkling ntawm daim tawv nqaij thiabaccelerating daim tawv nqaij laus.6,7 Yog li ntawd, kev kho mob antioxidant kom tshem tawmROS thiab ua kom muaj peev xwm mitochondrial muaj qhov zoo los tiv thaiv daim tawv nqaij los ntawm kev laus.

Ntxiv rau ROS, elastase muaj lub luag haujlwm tseem ceeb hauv kev poobdaim tawv nqaij elasticity, uas rhuav tshem elastin, ib qho tseem ceeb ntawm cov proteinextracellular matrix.8 Elastin, vim nws qhov tseem ceeb elastic recoilCov yam ntxwv, muab elasticity rau daim tawv nqaij, whereas elastase muajmuaj peev xwm tshem tawm elastin thiab lwm yam proteins.8 Yog li ntawd, inhibition ntawm elastase enzyme tuaj yeem yog lub tswv yim tseem ceeb rau daim tawv nqaij sagging ntawm tiv thaiv qhov poob ntawm daim tawv nqaij elasticity.

Hauv txoj kev tshawb no, peb tau tsim cov khoom siv tshiab uas sib xyaw ua keperoxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1)a) - tau txais peptide TPPTTP thiab gallic acid (GA)siv rau hauv tshuaj pleev ib ce los tiv thaiv kev laus. GA yog ib qho phytochemical pom nyob rau hauv ntau yam txiv hmab txiv ntoo thiab zaub, nrog rau txiv hmab txiv ntoo, txiv lws suav, thiab ntsuab tshuaj yej, uas paub tias muaj siab antioxidant thiab antibacterial. eua ffects.9 Txawm hais tias GA yog siv rau hauv cov tshuaj pleev ib ce thiab khoom noj khoom haus kev lag luam vim cov txiaj ntsig eua ffects, nws cov formulation nyhav tsis ruaj khov.10 Peb li tsim (galloyyl)2–KTPTP (Gal2–Pep) txhawm rau txhawm rau txhim kho kev ruaj ntseg ntawm GA los ntawm kev khi nws rau PGC-1a- tau txais peptide TTPTTP.

Hauv qhov kev tshawb fawb no, peb tau tsim ib qho (galloyl)2–KTPTP (Gal2– Pep) peptide formulation nyob rau hauv conjugation nrog gallic acid thiab paub tseeb tiasnws lub peev xwm ua antioxidant thiab antiaging tus neeg sawv cev.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Khoom siv

Dulbecco's ModifiedEagle's Medium (DMEM), penicillin / streptomycin, fetal bovine serum (FBS), phosphate-buua ffua eredsaline (PBS), thiab trypsin tau yuav los ntawm Gibco (Carls

yawg, CA). Hydroxybenzotriazole (HOBt), diisopropyl carbodiimide (DIC), 1, 8-diazabicyclo[5.4.0]undec-7-en (DBU),N, N- diisopropylethylamine (DIEA), gallic acid thiabN-succinyl-tri-alanyl-pnitroanilide tau txais los ntawm Sigma-Aldrich (St. Louis, MO).L-Form amino acids (Fmoc-Lys (Boc))–OH, Fmoc-Thr (tBu)–OH, thiab Fmoc-Pro–OH) tau yuav los ntawm Bead-Tech (Seoul,Kauslim).

2.2. Synthesis ntawm peptides thiab gallic acid-coupled peptides

Tag nrho cov peptides tau tsim los ntawm 2-chlorotrityl chloride resin (200mmol scale, hloov tus nqi¼ 1,46 mmol g 1) siv HOBt–DIC mediated solid-phase peptide synthesis (SPPS).

Peptide synthesis siv SPPS txoj kev tau ua nrog me ntsis modications hais txog cov kev tshawb fawb yav dhau los.11–13 Lub 2-chlorotrityl chloride (CTC) resin tau swelled hauv dichloro

methane (DCM, 8 mL) rau 30 min. Cov resin tau ntxuav nrogDimethylformamide (DMF). Tag nrho cov tshuaj tiv thaiv tau ua nyob rau hauv chav tsev kub. Fmoc tiv thaiv amino acid derivatives (2 sib npaug(Equiv.), for thefi uarst txuas amino acid lossis 5 equiv., rau lwm cov amino acids) tau ua ke nrog DIEA (5 equiv., rau ib qho amino acid txuas) lossis HOBt / DIC (6 equiv./5 equiv.) hauv DMF aftuaua qhov kev tiv thaiv ntawm Fmoc siv 2 feem pua ​​(v/v) DBU hauv DMF (2 zaug, 2 min ib zaug, 8 mL). Thiab tag nrho cov kauj ruam tau ntxuav 5 zaug nrog DMF. Thaum kawg gallic acid tau ua ke nrog lysine-txuas peptides.Tom qab kev sib txuas ntawm gallic acid, cov resin yogantxuav, ntxuav,thiab qhuav hauv qab lub tshuab nqus tsev siab. Cov tshuaj cleavage (TFA:deionized [DI] dej: TIS¼ 95 : 2.5 : 2.5, v/v/v feem pua ​​) raug kho rau 2 hkom tau cov crude peptides. Cov peptides crude tau precipitated thiab ntxuav peb zaug nrog txias diethyl ether.

Analytical rov qab-theem high-performance kua chromatography(RP-HPLC) tau ua nyob rau ntawm Dej 2695 Sib caisModule nrog Capcell Pak C18 kem (4.6 hli 250 hli, 5mm, Shiseido). Lub sijhawm txawb muaj 0.05 feem pua ​​TFA hauv H2O (mobile theem A) thiab 0.05 feem pua ​​TFA hauv acetonitrile (mobile theem B).

Elution tau ua tiav los ntawm kev siv ib qho linear gradient rau mobiletheem B ntawm 5 feem pua ​​​​rau 65 feem pua ​​​​ntawm 30 min ntawm aow tus nqi ntawm 1.0 mL min 1. Cov peptide peaks tau kuaj pom ntawm lub wavelength ntawm 230 nm. Semi-preparative RP-HPLC tau ua los ntawm Waters HPLC system (Twj 600E, Ntes UV-484) nrog Gemini RP-C18kab (21.2 mm 250 hli, 5mm, Phenomex). Lub sijhawm txawb muaj 0.05 feem pua ​​TFA hauv H2O (mobile theem A)thiab 0.05 feem pua ​​TFA hauv acetonitrile (mobile theem B). Elution tau ua tiav los ntawm kev ua haujlwm ib txoj kab gradient rau theem B ntawm mobile7% mus rau 22 feem pua ​​​​ntawm 15 min ntawm alow nqi 10 mL min 1. Covpeptide ncov tau kuaj pom spectrophotometrically ntawm lub wavelength ntawm 230 nm.

Cov peptides synthesized tau yaj nyob rau hauv 5 feem pua ​​​​ formic acid hauv dej thiab tshuaj xyuas ntawm electrospray ionization (ESI) /quadrupole-lub sijhawm-ntawm-light (Q-TOF) tandem loj spectroscopy(MS) system (TripleTOF6600, ABSciex, Foster City, CA). Cov qauv raug txhaj rau hauv Q-TOF system nruab nrognano-spray qhov chaw ntawm alua rpa1mL min 1. ESI ionQhov chaw tsis muaj raws li hauv qab no: ib qho hluav taws xob ion txau ntawm 1.5 kV, cov pa hluav taws xob ntawm 10 psi, thiab cov roj sheath ntawm 5 psi. Lub spectrum nyob rau hauvtag nrho-scan hom thiab MS / MS tau sau nyob rau hauv ntau yam ntawmm/z

100–1200 Da thaum lub sij hawm tsub zuj zuj ntawm 25 ms ib spectrum.Lub zog sib tsoo yog ramped ntawm 10 mus rau 80. Qhov tshwm simCov ntaub ntawv tau txais los ntawm Analyst TF yog likhoom thiab txiavmuab los ntawm centroid 80 feem pua ​​nrog qhov tsawg kawg nkaus ncov, suab nrov txo ntawm 10 feem pua, nruab nrab deisotoping nrog ib tug 3 feem pua, tsis muaj suab nrov txo, thiab tsis smoothing. Synthesized peptides tau txheeb xyuasnrog ib tug loj kam rau ua ntawm 0.05 Da thiab manually sequenced siv MS/MS kam rau ua ntawm 0.01 Da.

2.3. Cell kab lis kev cai thiab kev soj ntsuam kev muaj sia nyob

Tib neeg, neeg laus, tsis tshua muaj calcium, siab kub (HaCaT) hlwbthiab CCD-1064Sk ( tib neeg daim tawv nqaijfibroblast) hlwbkab lis kev cai hauv DMEM nrog 10 feem pua ​​FBS thiab 1 feem pua ​​penicillin /tshuaj streptomycin. Cov hlwb tau incubated ntawm 37 C nyob rau hauv ib tug humidified cua chamber nrog 5 feem pua ​​CO2.Rau qhov kev ntsuam xyuas muaj peev xwm, cov khoom siv ntawm tes suav -8 (CCK-8, Dojindo Co. Ltd. Beijing, Tuam Tshoj) tau siv. HaCaT thiab CCD-1064Sk cellstau noob hauv 96-zoo microplates (5 103 cell per well) thiab incubated ntawm 37 C hauv 5 feem pua ​​​​CO2 rau 24h. Cov kab lis kev cai tom qab ntawd raug nthuav tawm rau ntau qhov sib txawv ntawm cov peptides los yog GA thiab incubated ntawm 37 C hauv 5 feem pua ​​​​CO2 rau 24h. Cell viabilitytau ntsuas tom qab siv CCK-8 raws li cov chaw tsim khoom cov lus qhia.

2.4. Kev txiav txim siab ntawm kev ua haujlwm antioxidant

2.4.1. DPPH kev xeem.Kev ua haujlwm antioxidant ntawm synthesizedpeptides thiab GA raug soj ntsuam ib leeg siv DPPH assay. DPPH kev soj ntsuam tau ua raws li qhov tau tshaj tawm yav dhau los

methodology nrog me me modication.12,14–16 Ua ntej, ib qho 0.12 mg mL 1 DPPH tov tau npaj rau hauv methanol, ces 100mL ntawm DPPH tov thiab 100mL ntawm cov synthesized peptides lossis GA tau sib xyaw ntawm qhov sib txawv ({{0}}.1 mM, 0.05 mM, 0.01 mM, thiab 1mM) hauv 96-zoo microplates. Nyob rau hauv lub orbital co incubator, lubcov tshuaj sib tov tau tshwm sim rau 30 feeb ntawm 37 C thiab 100 rpm tsis muajteeb. Tom qab ntawd lub absorbance yog ntsuas ntawm 517 nm thiab lub peev xwm antioxidant raug xam.

2.4.2. ROS scavenging kev ua si.

Intracellular ROS scavenging kev ua si raug soj ntsuam siv 20, 70-dichlorofluorescein diacetate cov khoom siv(DCF-DA, Abcam, USA). HaCaT hlwb raug noob hauv 96-zoo microplates (5 103 cell per well) thiab incubated ntawm 37 C rau 24 h.After incubation, cov kab lis kev cai tau raug rau cov synthesized peptides los yog GA (100mM) thiab incubated ntawm 37 C rau 24 h. Tom qab ntawd cov hlwb tau nthuav tawm rau 10mM DCF-DA tov thiab incubated rau 30 min. After incubation, cov tshuaj tau ntxuav nrog PBS. Covcov hlwb tau txheeb xyuas siv tus nyeem ntawv microplate ntawm excitation thiab

emission wavelengths ntawm 485nm thiab 530nm, feem.

Daim duab 1Synthetic kauj ruam thiab ib theem zuj zus kev pom zoo ntawm peptides siv quadrupole-lub sij hawm ntawmya davhlau(Q-TOF) pawg spectrometry: (a) cov ncauj lus kom ntxaws synthetic txheej txheem (b) TPPTTP, (c) galloyl–TPPTTP, thiab (d) (galloyl)2–KTPPTTP.

2.5. Mitochondrial membrane muaj peev xwm soj ntsuam

JC-1 dye (Thermo Fisher, USA) tau siv rau mitochondrialKev ntsuam xyuas cov peev txheej membrane (MmP). CCD-1064Sk thiab HaCaT hlwb tau cog rau hauv 96-zoo microplates (5 10 3cell per well)

thiab incubated ntawm 37 C thiab 5% CO2 rau 24h. Qabyog incubation,Cov kab lis kev cai raug nthuav tawm ntau qhov sib txawv ntawm cov peptides synthesized thiab incubated ntawm 37 C hauv 5 feem pua ​​​​CO2 rau 24h.JC-1 dye tau ntxiv rau CCD-1064Sk thiab HaCaT hlwb rautsim ib qhov kawg concentration ntawm 10mM. After 10 min ntawm incubation, lub hlwb raug ntxuav ob zaug nrog 37 C PBS. Ntsuab thiab liabfluorescencetau ntsuas siv Varioskan LUXMultimode Microplate Reader (ThermoFisher, USA) ntawm excikev (lEx) / emission (lEm475/530 nm thiab alEx/lEmntawm 475 /590nm ,ua.

2.6. Elastase inhibitory kev ua ub no

CCD-1064Sk hlwb tau noob nyob rau hauv 6-cov phiaj zoo thiab incubated ntawm 37 C hauv 5 feem pua ​​​​CO2 rau 24h. Cov kab lis kev cai tom qab ntawd tau nthuav tawm mus rau qhov concentration ntawm 100mM ntawm cov synthesized peptides los yog GA thiab incubated ntawm 37 C hauv 5 feem pua ​​​​CO2 rau 24h. Cells raug ntxuav nrog dPBS ob zaug thiab peb tau sau txhua lub xov tooj siv xov tooj ntawm tes scraper. Aer ntxiv 0.2 M Tris–HCl (pH 8.0) nrog 0.1 feem pua ​​Triton-X rau cov sauhlwb, hlwb raug homogenized los ntawm ultrasonicator. Cov tshuaj homogenized yog centrifuged rau 20 min ntawm 3000 rpmthiab 4 C. Ces, after noj cov supernatant, protein ntau-cation tau ua los ntawm kev kuaj BCA protein. Protein rau txhua tusqauv tau muab xa mus rau hauvfi uaconcentration ntawm 100mgmL ib 1, thiabN-succinyl-tri-alanyl-p- nitroanilide tau ntxiv rau ntawmQhov kawg concentration ntawm 1.6 mM thiab reacted ntawm 36 C rau 1 h.Tom qab ntawd, qhov nqus tau raug ntsuas ntawm 405 nm siv tus nyeem ntawv microplate.

2.7. RT-qPCR

Cov qib mRNA ntawm cov cim tiv thaiv kev laus hauv CCK-1064Sk hlwbtau soj ntsuam siv RT-qPCR. Tag Nrho RNA tau sau siv TRIzol reagent (Life Technologies, Carlsbad, CA) thiab thim rov qab

tau sau tseg siv PrimeScript RT reagent kit (Takara BioInc., Kusatsu, Nyiv). CFX96 system (Bio-Rad Laboratories, Hercules, CA) thiab iQ SYBR Green Supermix (Bio-Rad Labora

tories) tau siv rau RT-qPCR.b-Actin tau siv los normalize mRNA qib ntawm hom I collagen, MMP-1, thiab PGC-1a. 

2.8. Kev txheeb cais

 Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab standard deviation ntawmpeb qhov kev ntsuas kev ywj pheej thiab tshuaj xyuas siv Cov Tub Ntxhais Kawmt-tests, nrog ap < 0.05 considered to be a signitsis txawv.

Nug ntxiv:

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