Tshooj 2 Antioxidation Thiab Cytoprotection Ntawm Acteoside Thiab Nws Cov Derivatives: Kev Sib Piv Thiab Mechanistic Chemistry
Mar 08, 2022
Tshooj 2 Cistanche Acteoside antioxidant tiv thaiv cov hlwb li cas?
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Cistanche deserticola muaj ntau yam teebmeem, nyem qhov no kom paub ntau ntxiv
3. Kev sib tham
Cov tshuaj tiv thaiv antioxidant ntawm ntuj phenolic tebchaw paub tias muaj kev koom tes hauv kev hloov hluav taws xob (ET) [18,19]. Yog li, qee qhov ET-raws li kev ntsuas hlau txo tau siv dav los ntsuas cov tshuaj tiv thaiv antioxidant ntawm phenolics, xws li FRAP thiab CUPRAC kev soj ntsuam. FRAP cov lus qhia yuav tsum ua kom tiav nrog pH tsawg dua 3.6. Xws li ib puag ncig acidic tau ntse suppressed H ntxiv ionization los ntawm phenolics; Yog li, FRAP kev soj ntsuam yog suav tias yog ib qho txheej txheem ET [20,21]. Kev ua tau zoo ntawmacteosidethiab nws cov derivatives nyob rau hauv qhov kev ntsuam xyuas FRAP txhais tau hais tias, thaum acteoside thiab nws cov derivatives ua antioxidants, lawv tuaj yeem siv txoj hauv kev ET los tawm tsam lawv cov tshuaj tiv thaiv antioxidant.
Tsis tas li ntawd, peb kuj tau ua qhov kev ntsuam xyuas CUPRAC hauv pH 7.4 tsis nyob. Raws li pom hauv Suppl. 1,acteosidethiab nws cov derivatives koob tshuaj-raws li nce lawv Cu2 plus- txo qis zog feem pua, qhia tias lawv tuaj yeem nyob twj ywm ET lub peev xwm ntawm lub cev pH. Txawm li cas los xij, lawv cov peev txheej ET poob qis hauv qhov kev txiav txim hauv qab no:acteoside> forsythoside B > polyumoside (Table 1). Qhov kev hloov pauv no qhia meej tias apiosyl moiety hauv forsythoside B thiab rhamnosyl moiety hauv poliumoside txo qis ET peev xwm.
Txhawm rau kuaj qhov ua tau tias ET tshwm sim thaum lawv cov txheej txheem radical-scavenging, oxygen-centered dawb radical PTIO ・ tau qhia hauv txoj kev tshawb no. Cyclic voltammetry pov thawj tau qhia tias PTIO・-scavenging hauv qab pH 5.0 yog ib qho tshuaj tiv thaiv electron-redox. Qhov kev soj ntsuam ntawdacteosidethiab nws cov derivatives tuaj yeem tshem tawm PTIO ・ radical ntawm pH 4.5, qhia txog qhov muaj peev xwm ntawm ET thaum lawv cov txheej txheem radical-scavenging. Pom tseeb, qhov kev tshawb pom no txhawb ntxiv cov txiaj ntsig tau hais los ntawm FRAP thiab CUPRAC kev soj ntsuam, thiab cov txiaj ntsig yav dhau los uas pub dawb electron (e) yog ib qho ntawm phenolic antioxidants [23].
Ntawm physiological pH 7.4, txawm li cas los xij, PTIO ・-scavenging essay tsis yog ib txoj hauv kev ET nkaus xwb tab sis kuj suav nrog txoj hauv kev hloov pauv proton- (H ntxiv). Thaum lub sij hawm tus txheej txheem, PTIO ・ tau raug pom zoo kom txais Hntxivlos ntawm phenolics los tsim cov khoom siab tshaj ([PTIO-H]ntxiv). Vim Hntxiv-hloov ib txwm nrog ET hauv stepwise lossis synchronous mechanisms [24], qhov tseeb (lossis kawg) cov khoom yog [PTIO-H] molecule [22]. PTIO・-scavening ntawm pH 7.4 (Suppl. 1) txhais tau tiasacteosidethiab nws cov derivatives muaj H ntxiv -hloov muaj peev xwm thiab. IC50 qhov tseem ceeb (Table 1) qhia tias tus txheeb ze Hntxiv-hloov muaj peev xwm nyob rau hauv nqe lus txiav txim ntawmacteoside>forsythoside B > polyumoside. Kom meej meej, cov apiosyl thiab rhamnosyl moieties kuj tsis muaj zog Hntxiv-hloov muaj peev xwm thaum lub sij hawm cov txheej txheem antioxidant.
Raws li yav tas los tham, thaum lub sij hawm antioxidant txheej txheem ntawm phenolics, ET feem ntau yog nrog proton (H.ntxiv) hloov mus ua ntau yam tshuaj tiv thaiv kab mob antioxidant [24], xws li hydrogen-atom hloov chaw (HAT) [23,25-27], sequential electron-proton hloov (SEPT) [26,27], sequential proton poob ib-electron hloov pauv (SPLET) [26], thiab kev hloov pauv hluav taws xob proton-coupled (PCET) [24-26,28]. Piv txwv li, ABTS plus • -scavenging, ib qho tshuaj tiv thaiv los ntawm ib leeg-electron hloov chaw (SET) [29], kuj tau raug pov thawj tias muaj kev cuam tshuam los ntawm H ntxiv rau qib tsis ntev los no [30]. ABTS ntxiv • -scavenging yog li ntawd muaj ntau txoj hauv kev-raws li tshuaj tiv thaiv antioxidant [21,31]. Qhov tseeb tiasacteosidethiab nws cov derivatives tuaj yeem tshem tawm ABTS ntxiv ・ radicals qhia tias lawv cov tshuaj tiv thaiv antioxidant kuj tseem tuaj yeem kho los ntawm ntau txoj hauv kev. Qhov kev xav no tau lees paub ntxiv los ntawm cov pov thawj los ntawm DPPH・-scavenging assay, cov tshuaj tiv thaiv muaj HAT, ET, SEPT, thiab PCET ntau txoj hauv kev [26,32]. Txawm li cas los xij, kev txheeb xyuas qhov ntau raws li IC50qhov tseem ceeb (Table 1) qhia tias nyob rau hauv ntau txoj hauv kev-raws li ABTS ntxiv rau --scavenging thiab DPPH*-scavenging yam,acteosideyog superior rau nws apposite forsythoside B thiab rhamnoside poliumoside. Yog li, nws tuaj yeem txiav txim siab tias apiol thiab rhamnosyl moieties nws thiaj li cuam tshuam ntau txoj hauv kev muaj peev xwm (tshwj xeeb yog ET thiab H.ntxiv-hloov) thaum lub sijhawm dawb-radical-scavening txheej txheem.
Raws li tau sau tseg los ntawm cov kws sau ntawv thiab lwm tus [14,26], thaum lub sijhawm ua haujlwm antioxidant, RAF cov tshuaj tiv thaiv kuj tseem tshwm sim. Txhawm rau txheeb xyuas qhov muaj peev xwm RAF, txawm li cas los xij, peb phenylpropanoid glycosides nrog rau caffeic acid tau kawm siv UPLC-ESI-Q-TOF-MS / MS tsom xam. Caffeic acid tau pom los ua cov khoom lag luam dimer, thaum peb phenylpropanoid glycosides ua tsis muaj qhov siab tshaj ntawm RAF cov khoom. Qhov kev tshawb pom no qhia meej tias peb phenylpropanoid glycosides tsis tuaj yeem ua raws li RAF txoj hauv kev los tawm tsam lawv cov tshuaj tiv thaiv antioxidant. Txij li thaum peb phenylpropanoid glycosides tuaj yeem suav tias yog cov esters ntawm caffeic acid (Daim duab 1), xws li qhov sib txawv ntawm caffeic acid thiab caffeic acid esters kuj qhia tau tias moiety loj tuaj yeem cuam tshuam cov tiam ntawm RAF.
Ua ke, los ntawm ib qho dawb-radical-scavening nam,acteosidethiab nws cov derivatives yuav dhau mus rau ntau txoj hauv kev los tawm tsam lawv cov tshuaj antioxidant. Cov kab mob antioxidant tsawg kawg yog koom nrog ET thiab H ntxiv -hloov (tab sis tsis yog RAF). Peb qhov kev tshawb pom yog ib feem txhawb los ntawm kev kawm theoretical uasacteosidetuaj yeem ua haujlwm antioxidant ntawm txoj kev SPLET. Hauv cov txheej txheem, acteoside tuaj yeem ua ntej deprotonate (Hntxiv-hloov) kom tawm los anion. Lub deprotonation yog xav tias yuav tshwm sim nyob rau hauv catechool moieties nrog qaug zog acidity. Tom qab ntawd, cov anion pub dawb electrons los muab phenoxy radical daim ntawv [33]. Phenoxy radicals nrog pn conjugation txawm li cas los xij yog ruaj khov rau qee qhov. Tau kawg, hauv qhov kev hwm no, kev sim ua haujlwm ntxiv yog xav tau yav tom ntej.
Nws yog tsim nyog hais tias cellular oxidative kev nyuaj siab kuj tuaj yeem tshwm sim los ntawm cov hlau hloov pauv (tshwj xeeb tshaj yog Fe.2 plus). Lub Fe2 plusion, txawm li cas los xij, tuaj yeem hloov H2O2molecule rau hauv qhov teeb meem tshaj plaws •OH radicals ntawm Fenton cov tshuaj tiv thaiv (Fe2Ntxiv rau H2O2 T Fe3plus plus ・OH plus OH-). Yog li ntawd, attenuation ntawm Fe2Cov qib ntxiv tuaj yeem cuam tshuam zoo ・ OH radicals tso tawm cellular oxidative kev nyuaj siab. Qhov tseeb, hlau-chelating los ntawm ntuj phenolic antioxidants tam sim no tau tsim los rau hauv kev kho mob zoo rau qee yam kab mob oxidative-kev nyuaj siab [34,35].
Hauv kev tshawb fawb tam sim no, acteoside thiab nws cov derivatives tau pom tias ua tau zoo Fe2 plus-chelators los ntawm kev hloov pauv hauv spectroscopy thiab cov xim daws teeb meem (Daim duab 2). Txawm li cas los xij, acteoside yog qhov qis dua ob lub glucosides hauv chelating Fe2 plusthiab forsythoside B nrog apiosyl moiety yog qis dua rau poliumoside nrog rhamnosyl moiety. Raws li kev sib piv ntawm lawv qhov kev nyiam sib haum xeeb (Daim duab 1, txoj cai), nws tau hais tias apiosyl (los yog rhamnosyl) moiety tuaj yeem pab cov ligand (phenylpropanoid pawg) hauv chelating Fe.2 plus. Xws li cov nyhuv synergistic undoubtedly ntxiv dag zog rau Fe2 plus-chelating muaj peev xwm thiab ua kom cov UV-vis peaks. Txawm li cas los xij, rhamnosyl muaj txiaj ntsig zoo dua li apiosyl hauv nws cov Fe2 plus - chelating peev xwm. Qhov sib txawv tuaj yeem raug ntaus nqi los ntawm qhov tseeb tias rhamnosyl tshwm sim hauv daim ntawv exocyclic (ie, aL-rhamnopyranosyl), thaum apiosyl nyob hauv daim ntawv pentacyclic (ie, pD-apiofuransyl). Ib daim ntawv exocyclic paub tias loj dua thiab ruaj khov dua. Yog li, exocyclic rhamnosyl yog qhov zoo dua li piv rau pentacyclic apiosyl hauv nws cov Fe.2 plus- chelating peev xwm.
Txhawm rau kuaj seb acteoside thiab nws cov txiaj ntsig tuaj yeem rub tawm ROS, peb tau ua qhov kev ntsuam xyuas pyrogallol autoxidation. Raws li pom hauv Suppl. 1, txhua tus tuaj yeem tshem tawm cov - radical, ib yam ROS tshwm sim hauv hlwb. Txawm li cas los xij, qhov txheeb ze bioactivity txo qis hauv qhov kev txiav txim poliumoside> forsythoside B>acteoside. Qhov kev txiav txim no kuj zoo ib yam rau cov teebmeem cytoprotective (Table 2). Qhov kev tshawb pom no qhia tau hais tias cov txiaj ntsig ntawm rhamnosyl moiety lossis apiosyl moiety yog txhawm rau txhim kho ROS-scavenging lossis cytoprotective teebmeem.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Tshuaj thiab Tsiaj
Acteoside (CAS naj npawb: 61276-17-3, 97%), forsythoside B (CAS naj npawb: 81525-13-5,97%) tau txais los ntawm BioBioPha (Kunming, Tuam Tshoj, Suppl. 3). Poliumoside (CAS naj npawb: 94079-81-9, 97 feem pua ) raug cais los ntawm peb pab neeg los ntawm cov tshuaj suav tshuajCallicarpa ibHT Chang (Suppl. 3). DPPH・,(±)-6-hydroxyl-2,5,7,8-tetramethlychromane-2-carboxylic acid (Trolox), 2,9-dimethyl{{ 9}}, 10-phenanthroline (neocuproine), 2,4, 6-tripyridyltriazine (TPTZ), thiab pyrogallol tau yuav los ntawm Sigma-Aldrich Shanghai Trading Co. (Shanghai, Suav). (NHq'ABTS [2,2'-azino-bis (3-ethylbenzene-thiazoline-6-sulfonic acid diammonium ntsev)] tau los ntawm Amresco Chemical Co. (Solon, OH, USA). PTIO・radical tau yuav los ntawm TCI Development Co., Ltd. (Shanghai, Suav teb) Caffeic acid tau yuav los ntawm National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). (FBS), thiab trypsin tau yuav los ntawm Gibco (Grand Island, NY, USA). AnnexinV/propidium iodide (PI) assay kit yog muas los ntawm Invitrogen (Carlsbad, CA, USA).
Sprague-Dawley (SD) nas ntawm 4 lub lis piam hnub nyoog tau txais los ntawm Tsiaj Center ntawm Guangzhou University of Chinese Medicine. Cov txheej txheem ntawm qhov kev sim no tau ua nyob rau hauv kev saib xyuas ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees Tsiaj hauv Guangzhou University of Suav (pom zoo 20170306A).
4.2. Metal-Reduction Assays (FRAP& CUPRAC)
Cov kev ntsuas hlau txo qis suav nrog Fe3 plus ib-txo lub zog ntsuas thiab Cu2 plus- txo hwj chim ntsuas. Lub Fe3 plus ib-txo kev ntsuam xyuas tau tsim los ntawm Benzie thiab Strain thiab raug hu ua FRAP [20]. Cov txheej txheem kev sim ntawm qhov kev ntsuas no tau piav qhia hauv tsab ntawv tshaj tawm dhau los [9]. Luv luv, FRAP reagent tau npaj tshiab los ntawm kev sib xyaw 10 mM TPTZ, 20 mM FeCl3,thiab {{0}}.25 M acetate tsis nyob ntawm qhov piv ntawm 1:1:10 ntawm pH 3.6. Cov qauv kuaj (x=4-20 L, 0.05 mg / mL) tau ntxiv rau (20 — x) ntawm 95 feem pua ethanol ua raws li 80 RL ntawm FRAP reagent. Tom qab 30-min incubation ntawm ambient kub, lub absorbance yog ntsuas ntawm 595 nm siv ib tug microplate nyeem ntawv (Multiskan FC, Thermo Scientific, Shanghai, Suav teb). Tus txheeb ze txo hwj chim ntawm tus qauv yog xam los ntawm cov qauv hauv qab no:
qhov twg Amaxyog qhov siab tshaj plaws absorbance ntawm cov tshuaj tiv thaiv sib tov nrog cov qauv, thiab Aminyog qhov tsawg kawg nkaus absorbance hauv kev sim. A yog qhov nqus ntawm cov qauv.
Cu2 plus-txo lub zog tuaj yeem ua tus yam ntxwv ntawm qib antioxidant thiab yog li hu ua CUPRAC. Qhov kev ntsuam xyuas no tau ua raws li ib txoj kev luam tawm yav dhau los [36]. Luv luv, 12 RL ntawm CuSOq aqueous tov (10 mmol / L), 12 RL ntawm neocuproine ethanolic kua (7.5 mmol / L) thiab (75 — x) RL ntawm CH3COONH4buffer tov ({{0}}.1 mol/L, pH 7.5) tau muab ntxiv rau cov qhov dej uas muaj qhov sib txawv ntawm cov qauv (0.05 mg/mL, 4-20 |^L). Lub absorbance ntawm 450 nm tom qab 30 min tau ntsuas los ntawm kev nyeem cov lus hais saum toj no. Tus txheeb ze CUPRAC lub zog tau suav nrog cov qauv rau FRAP. Amaxyog qhov siab tshaj plaws absorbance ntawm cov tshuaj tiv thaiv sib tov nrog cov qauv, thiab Aminyog qhov tsawg kawg nkaus absorbance hauv kev sim. A yog qhov nqus ntawm cov qauv.
4.3. PTI 0・-ScaveningAssay
PTIO*-scavenging assays (ntawm pH 4.5 lossis pH 7.4) tau ua raws li peb txoj kev [16]. Hauv luv luv, cov qauv ntsuas ntsuas (x=0-20} ^L, 1 mg / mL rau pH 4.5 thiab {{10}}.5 mg / mL rau pH 7.4) tau ntxiv rau (20 — x) rL ntawm 95 feem pua ethanol, ua raws li 80 RL ntawm aqueous PTIO * daws. Cov tshuaj aqueous PTIO * tau npaj siv cov tshuaj phosphate-butter (0.1 mM, pH 4.5 lossis pH 7.4). Qhov sib tov tau khaws cia ntawm 37 degree rau 2 h, thiab tom qab ntawd ntsuas qhov nqus ntawm 560 nm siv cov lus hais saum toj no microplate nyeem ntawv. Qhov feem pua PIO * inhibition tau xam raws li hauv qab no:
qhov twg A degree yog qhov absorbance ntawm kev tswj tsis muaj tus qauv, thiab A yog qhov absorbance ntawm cov tshuaj tiv thaiv sib xyaw nrog cov qauv.
4.4. ABTSntxiv*-Scaving thiab DPPH*- Kev Tshawb Fawb Kev Tshawb Fawb
ABTS*ntxiv-scavenging kev ua tau raug soj ntsuam raws li txoj kev [37]. ABTS plus * tau tsim los ntawm kev sib xyaw ntawm 0.2 mL ntawm ABTS diammonium ntsev (7.4 mmol / L) nrog 0.2 mL ntawm poov tshuaj persulfate (2.6 mmol / L). Cov sib tov tau khaws cia nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub rau 12 h kom tso cai rau ua tiav cov radical tiam ua ntej yuav diluted nrog distilled dej (ntawm ib tug ratio ntawm kwv yees li 1:20) kom nws absorbance ntawm 734 nm yog { {16}}.35 土 0.01 siv tus nyeem ntawv hais txog microplate. Txhawm rau txiav txim siab txog kev khawb av, cov qauv ntsuas (x=4-20 RL, 0.05 mg / mL) tau ntxiv rau (20 — x) RL ntawm cov dej distilled tom qab 80 RL ntawm ABTS ntxiv * reagent, thiab absorbance ntawm 734 nm tau ntsuas 3 min tom qab pib sib tov, siv cov dej distilled li qhov khoob.
DPPH * radical-scavenging kev ua haujlwm tau txiav txim siab raws li tau piav qhia yav dhau los [18]. Luv luv, 75 RL ntawm DPPH * tov (0.1 rM) yog tov nrog cov qhia concentrations ntawm tus qauv (0.025 mg/mL, 5-25 RL) yaj nyob rau hauv methanol. Qhov sib tov tau khaws cia ntawm chav sov li 30 min, thiab qhov nqus tau ntsuas ntawm 519 nm siv cov lus hais saum toj no microplate nyeem.
Qhov feem pua ntawm ABTS ntxiv rau •-scavenging kev ua si thiab DPPH*-scavenging kev ua si raug xam raws li cov qauv qhia hauv Tshooj 4.3.
4.5. UPLC—ESI-Q-TSO—MS/MS Analysis ntawm PPH* Cov khoom siv tshuaj tiv thaiv
Txoj kev no yog raws li peb txoj kev tshawb fawb yav dhau los [25]. Cov tshuaj methanol ntawm acteoside tau tov nrog kev daws ntawm DPPH * radicals hauv methanol ntawm qhov sib piv ntawm molar ntawm 1: 2, thiab cov txiaj ntsig sib xyaw yog incubated rau 24 teev ntawm chav tsev kub. Cov khoom sib tov ces lim los ntawm 0.22-Rm lim thiab tshuaj xyuas siv UPLC system nruab nrog C18 kem (2.0 mm id x 100 mm, 1.6 Rm, Phenomenex, Torrance, CA, USA). Lub sijhawm txawb tau siv rau qhov kev hloov pauv ntawm qhov system thiab muaj kev sib xyaw ntawm methanol (theem A) thiab dej (theem B). Kem tau eluted ntawm tus nqi ntws ntawm 0.3 mL / min nrog cov kev pab cuam gradient elution hauv qab no: 0-10 min, 60-100 feem pua A; 10-15 min, 100 feem pua A. Cov qauv txhaj tshuaj tau teem rau ntawm 1 RL rau kev sib cais ntawm cov khoom sib txawv. ESI-Q-TOF-MS / MS tsom xam tau ua tiav siv Triple TOF 5600ntxivMass spectrometer (AB SCIEX, Framingham, MA, USA) nruab nrog ESI qhov chaw, uas tau khiav hauv hom tsis zoo ionization. Qhov scan ntau yog teem rau ntawm 100-2000 Da. Lub kaw lus tau khiav nrog cov kev txwv hauv qab no: ion spray voltage, -4500 V; ion qhov cua sov, 550 degree C; curtain gas (CUR, N2), 30 psi; nebulizing roj (GS1, Cua), 50 psi; Tis gas (GS2, Air), 50 psi. Lub peev xwm txo qis (DP) tau teeb tsa ntawm -100 V, thaum lub zog sib tsoo (CE) tau teeb tsa ntawm -40 V nrog lub zog sib tsoo (CES) ntawm 20 V. Cov khoom RAF
tau txiav txim siab los ntawm kev rho tawm cov mis molecular los ntawm tag nrho cov ion chromatogram (Suppl. 2).
Cov kev sim hais saum toj no tau rov ua dua siv forsythoside B, podium sab, thiab caffeic acid. Qhov sib thoojm/zPeaks tau muab rho tawm los ntawm cov mis mos molecular los ntawm tag nrho cov ion chromatogram (Suppl. 2).
4.6. UV-Vis-Spectra Analysis ofFe2 plus- Cov khoom siv hluav taws xob
Lub Fe2 plus-Chelating cov tshuaj tiv thaiv cov khoom ntawm acteoside-Fe2 plustau soj ntsuam siv UV-Vis-spectroscopy [13]. Rau qhov kev sim, 300 ntawm cov tshuaj methanolic ntawm acteoside (0.24 mM) tau ntxiv rau 700 rL ntawm cov tshuaj aqueous ntawm FeCl? 4H2O (168 mM). Txoj kev daws yog ces sib xyaw ua ke. Tom qab ntawd, qhov sib xyaw ua ke tau raug tshuaj xyuas siv UV-Vis spectrophotometer tom qab ib teev (Unico 2600A, Shanghai, Tuam Tshoj) los ntawm 200-850 nm.
Cov kev sim hais saum toj no tau rov ua dua siv forsythoside B, lossis podium sab, tsis yog acteoside.
4.7. Pyrogallol Autooxidation Assay rau Superoxide Anion (•O2~) Kev khawb
Kev ntsuas ntawm superoxide anion (・.{{0}}) kev ua si pov tseg yog raws li peb txoj kev [17]. Luv luv, cov qauv tau yaj hauv ethanol ntawm 1 mg / mL. Cov qauv tshuaj (x rL) tau tov nrog Tris-HCl tsis (980 — x rL, 0.05 M, pH 7.4) uas muaj EDTA (1 mM). Thaum 20 RL pyrogallol (60 mM hauv 1 mM HCl) tau ntxiv, qhov sib tov tau shaken ntawm chav sov tam sim ntawd. Lub absorbance ntawm 325 nm ntawm qhov sib tov tau ntsuas (Unico 2100, Shanghai, Tuam Tshoj) tiv thaiv Tris-HCl tsis raws li qhov khoob txhua 30 s rau 5 min. Lub peev xwm scavenging tau xam raws li hauv qab no:
4.8. Cytoprotective Effects Towards Oxidatively Stressed bmMSCs (MTT Assay)
Cov bmMSCs tau raug coj los ua raws li peb cov ntaub ntawv dhau los [38] nrog kev hloov kho me ntsis. Hauv luv luv, pob txha pob txha tau txais los ntawm femur thiab tibia ntawm tus nas. Cov qog nqaij hlav tau diluted nrog DMEM (tsawg qab zib) uas muaj 10 feem pua FBS. Cov bmMSCs tau npaj los ntawm gradient centrifugation ntawm 900 g rau 30 min ntawm 1.073 g / mL Percoll. Cov hlwb npaj tau raug tshem tawm los ntawm kev kho mob nrog 0.25 feem pua trypsin thiab dhau mus rau hauv cov kab lis kev cai ntawm 1 x 104 / cm2. Cov bmMSCs ntawm nqe 3 tau raug soj ntsuam rau kev coj noj coj ua ntawm cov cell homogeneity siv kev tshawb nrhiav CD44 siv MTT assay [39].
Qhov kev ntsuam xyuas MTT tau siv los ntsuas cov txiaj ntsig cytoprotective ntawm acteoside thiab nws cov txiaj ntsig ntawm bmMSCs [40]. Tus qauv raug mob raug tsim los ntawm kev tshawb fawb yav dhau los [41]. Cov txheej txheem kev sim tau piav qhia luv luv hauv daim duab 3.
4.9. Kev txheeb cais
Txhua qhov kev sim hauv Tshooj 4.2-4.4 thiab 4.7 tau ua nyob rau hauv triplicate, thiab MTT kev sim ntsuas tau ua hauv pentaplicate. Cov ntaub ntawv raug kaw raws li qhov nruab nrab土SD (tus qauv sib txawv). Cov koob tshuaj tiv thaiv kab mob tau npaj siv Keeb Kwm 6.0 software tshaj lij (OriginLab, Northampton, MA, USA). Tus nqi IC50 tau txhais tias yog qhov kawg siab ntawm 50 feem pua radical inhibition (tus txheeb ze txo hwj chim) [42]. Kev sib piv kev txheeb cais tau ua los ntawm ib txoj kev ANOVA txhawm rau txheeb xyuas qhov sib txawv tseem ceeb siv SPSS 13.0 (SPSS Inc., Chicago, IL, USA) rau Windows.p< 0.05 raug suav hais tias yog qhov tseem ceeb ntawm kev txheeb cais.
5. Cov lus xaus
Peb lub ntuj phenylpropanoid glycosides, uas yog, acteoside, forsythoside B, thiab podium sab, tuaj yeem koom nrog ntau txoj hauv kev los ua kom muaj zog tiv thaiv antioxidant, suav nrog ET, H ntxiv -hloov; thiab Fe2 plus-chelating, tab sis tsis RAF. ET thiab Hntxiv-Txoj kev hloov pauv yuav raug cuam tshuam los ntawm rhamnosyl moiety lossis apiosyl moiety; Txawm li cas los xij, Fe2 plus-chelating txoj kev tuaj yeem txhim kho los ntawm cov piam thaj residues (tshwj xeeb tshaj yog rhamnosyl moiety). Cov txiaj ntsig dav dav ntawm rhamnosyl moiety lossis apiosyl moiety yog txhawm rau txhim kho ntau txoj hauv kev cuam tshuam nrog ROS-scavenging peev xwm. Yog li, forsythoside B thiab poliumoside zoo dua rau acteoside hauv cytoprotective teebmeem.
Cov khoom siv ntxiv:Cov ntaub ntawv ntxiv muaj nyob online. 1. koob tshuaj tiv thaiv kab mob; 2. HPLC-MS spectra; 3. Kev soj ntsuam daim ntawv pov thawj ntawm acteoside, forsythoside B, thiab poliumoside.
Kev lees paub:Txoj haujlwm no tau txhawb nqa los ntawm National Science Foundation ntawm Tuam Tshoj (81573558, 81603269), Guangdong Science thiab Technology Project (2017A050506043), thiab Natural Science Foundation ntawm Guangdong Province (2 017A030312009, 2015A03031049).
Tus sau kev koom tes:Xian Li thiab Dongfeng Chen xeeb thiab tsim cov kev sim; Aichi Wu npaj poliumoside; Yulu Xie, Qian Guo, thiab Penghui Xue tau ua cov kev sim tshuaj antioxidant; Ke Li thiab Wei Zhao tau ua qhov kev sim MTT; Hong Xie txheeb xyuas cov ntaub ntawv; Jiasong Guo ua qhov kev sim oi Daim duab 2D; Xian Li sau ntawv. Txhua tus kws sau ntawv tau nyeem thiab pom zoo cov ntawv sau kawg.
Kev tsis sib haum xeeb ntawm kev txaus siab:Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.
acteosidehauvcistanche
Cov ntawv luv
ABTS plus • 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) radical
bmMSCs pob txha pob txha-derived mesenchymal qia hlwb
CUPRAC cupric txo cov peev xwm antioxidant
dAMP 2'-deoxyadenosine-5'-monophosphate radical
DMEM Dulbecco's modified Eagle's medium
dGMP 2/-deoxyguanosine-5'-monophosphate radical
DPPHe 1, 1-diphenyl-2-picryl-hydrazine radical
ET hloov hluav taws xob; FBS: Fetal bovine serum
FRAP ferric ion txo cov antioxidant zog;
HAT hydrogen atom hloov pauv
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
PCET proton-coupled electron hloov
PTIOe 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical
RAF radical adduct tsim
ROS reactive oxygen hom
SCNT somatic cell nuclear hloov
SEPT sequential electron-proton hloov
SPLET sequential proton poob ib qho hluav taws xob hloov pauv
TPTZ 2,4,6-tripyridyl triazine
Trolox (±) -6-hydroxyl-2,5,7,8-tetramethlychromane-2-carboxylic acid
Cov ntaub ntawv
1. Kubica, P.; Zoopa, A.; Ekiert, H. Kev tsim cov verbascoside thiab phenolic acids nyob rau hauv biomass ntawm Verbena Officinalis L. (vervain) kab lis kev cai nyob rau hauv txawv nyob rau hauv vitro tej yam kev mob. Nat. Prod. Res. Xyoo 2017, 31, 1663-1668.
2. Lee, JH; Chun, JL; Kim, KJ; Kim, EJ; Kim, DH; Li, BM; Han, KW; Park, KS; Li, KB; Kim, MK Cov nyhuv ntawm Acteoside raws li Cell Protector los tsim ib tus dev Cloned. PLoS ONE 2016,11, e0159330.
3. Wang, HQ; Xu, YX; Yaj, J.; Zhao, XY; Sun, XB; Zhang, YP; Guo, JC; Zhu, CQ Acteoside Tiv Thaiv Tib Neeg Neuroblastoma SH-SY5Y Cells tiv thaiv |3-amyloid-induced Cell Injury. Lub hlwb Res. 2009, 1283, 139-147.
4. Yaj, JH; Yog, Y.; Liu, HB; Wang, JH; Hu, JP tiv thaiv cov teebmeem ntawm Acteoside tiv thaiv X-ray-induced puas nyob rau hauv tib neeg daim tawv nqaij Fibroblasts. Mol. Med. Rep. 2015,12, 2301-2306.
5. Liu, CH; Liu, TS; Luo, CQ; Zhang, J. Zeng, XY; Koj, L.; Xie, LJ Kev txiav txim siab ntawm forsythiaside B thiab poliumoside nyob rau hauv txawv keeb kwm thiab qhov chaw ntawm Callicarpa kwangtungensis. Zhongguo Zhong Yao Za Zhi 2013, 38, 3324-3326.
6. Jiang, WL; Tian, JW; Fu, FH; Zhu, HB; Hou, J. Neuroprotective Efficacy and Therapeutic Window of Forsythoside B: Ina Rat Model of Cerebral Ischemia and Reperfusion Injury. Eur. J. Pharmacol. Xyoo 2010, 640, 75-81.
7. Pan, N.; Hori, H. Antioxidant Action ntawm Acteoside thiab Nws Analogs ntawm Lipid Peroxidation. Redox Rep. 1996, 2, 149-154.
8. Zheng, RL; Shi, YM; Jia, ZJ; Zhao, CY; Zhang, Q.; Tan, XR Ceev Kho ntawm DNA Radicals. Chem. Soc. Rev. 2010, 39, 2827-2834.
9. Li, XC; Maiv, WQ; Chen, DF Chemical Study on Protective Effect Against Hydroxyl-induced DNA Damage and Antioxidant Mechanism of Myricitrin. J. Chin. Chem. Soc. Xyoo 2014, 61, 383-390.
10. Shi, YM; Wang, WF; Huang, CY; Jia, ZJ; Yao, S.; Zheng, RL Kho kho sai ntawm Oxidative DNA puas los ntawm Phenylpropanoid Glycosides thiab Lawv Cov Analogues. Mutagenesis 2008, 23, 19-26.
11. Jiang, Q.; Li, XC; Tian, YG; Lin, QQ; Xie, H.; Lu, WB; Chi, YG; Chen, DF Lyophilized Aqueous Extracts ntawm Mori Fructus thiab Mori Ramulus Tiv Thaiv Mesenchymal Stem Cells los ntawm eOH-kho Kev puas tsuaj: Bioassay thiab Antioxidant Mechanism. BMC Complement. Hloov. Med. Xyoo 2017, 17, 242.
12. Wang, TT; Zeng, GC; Li, XC; Zeng, HP In Vitro Studies ntawm Antioxidant thiab tiv thaiv nyhuv ntawm 2-Substituted-8-Hydroxyquinoline Derivatives tawm tsam H2O2-Induced Oxidative Stress hauv BMSCs. Chem. Biol. Drug Des. Xyoo 2010, 75, 214-222.
13. Liu, JJ; Li, XC; Lin, J.; Li, YR; Wang, TT; Jiang, Q.; Chen, DF Sarcandra Glabra (Caoshanhu) Tiv Thaiv Mesenchymal Stem Cells los ntawm Oxidative Stress: Kev Ntsuam Xyuas Bio thiab Mechanistic Chemistry. BMC Complement. Hloov. Med. Xyoo 2016, 16, 423.
14. Li, XC; Han, L.; Li, YR; Zhang, J. Chen, JM; Lu, WB; Zhao, XJ; Laj, YT; Chen, NWS; Wei, G. Cov nyhuv tiv thaiv ntawm Sinapine tiv thaiv Hydroxyl radical-induced puas rau Mesenchymal qia hlwb thiab muaj peev xwm siv tau. Chem. Pharm. Bull. Xyoo 2016, 64, 319-325.
15. Bertolo, A.; Kapossela, S.; Frankl, G.; Bau, M.; Potzel, T.; Stoyanov, J. Oxidative Status Predicts Quality in Human Mesenchymal Stem Cells. Stem Cell Res. Ther. Xyoo 2017, 8, 3.
16. Li, XC 2-Phenyl-4,4,5,5-Tetramethylimidazoline-1-oxyl 3-Oxide (PTIOe) Radical Scavenging: Ib Tug Tshiab thiab Yooj Yim Antioxidant Assay In Vitro. J. Agric. Khoom noj khoom haus Chem. Xyoo 2017, 65, 6288-6297.
17. Li, X. Kev Txhim Kho Pyrogallol Autoxidation Method: Ib qho kev ntseeg siab thiab pheej yig Superoxide-Scavenging Assay haum rau txhua yam tshuaj tiv thaiv antioxidant. J. Agric. Khoom noj khoom haus Chem. Xyoo 2012, 60, 6418-6424.
18. Li, XC; Jiang, Q.; Wang, TT; Liu, JJ; Chen, DF Kev sib piv ntawm cov tshuaj tiv thaiv antioxidant ntawm Quercitrin thiab Isoquercitrin: Nkag siab lub luag haujlwm ntawm 6〃-OH Pawg. Molecules 2016, 21,1246.
19. Leopoldini, M.; Marino, T.; Russo, N.; Toscano, M. Antioxidant Properties of Phenolic compounds: H-atom Versus Electron Transfer Mechanism. J. Phys. Chem. 2004, 108, 4916^922.
20. Benzie, IFF; Strain, JJ Lub Ferric Txo Peev Xwm ntawm Plasma (FRAP) raws li Kev Ntsuas ntawm "Antioxidant Fais Fab": FRAP kev soj ntsuam. Qhov quav. Biochem. Xyoo 1996, 239, 70-76.
21. Gulcin, I. Antioxidant Activity of Food Constituents: Ib Txheej txheem cej luam. Arch. Toxicol. 2012, 86, 345-391.
22. Goldstein, S.; Russo, UA; Samuni, A. Cov tshuaj tiv thaiv ntawm PTIO thiab Carboxy-PTIO nrog ・NO, ・NO2, thiab O2. J. Biol. Chem. Xyoo 2003, 278, 50949-50955.
23. Li, X.; Han, WJ; Maiv, WQ; Wang, L. Antioxidant Activity thiab Mechanism ntawm Tetrahydroamentoflavone hauv vitro. Nat. Prod. Pawg. Xyoo 2013, 8, 787-789.
24. Zhang, HY; Wu, W.; Mo, YR Txoj Kev Kawm ntawm Proton-Coupled Electron Transfer (PCET) nrog Plaub Lub Xeev Diabatic ntawm ab Initio Level. Tshuab xam zauv. Theor. Chem. Xyoo 2017, 1116, 50-58.
25. Lin, J.; Li, XC; Chen, L.; Lu, WZ; Chen, XWM; Han, L.; Chen, DF tiv thaiv cov nyhuv tiv thaiv Hydroxyl radical-induced DNA puas thiab Antioxidant Mechanism ntawm [6]-gingerol: Kev Tshawb Fawb Tshuaj. Bull. Korean Chem. Soc. Xyoo 2014, 35, 1633-1638.
26. Iuga, C.; Alvarez-Idaboy, JR; Russo, N. Antioxidant Kev Ua Haujlwm ntawm Trans-Resveratrol Mus Rau Hydroxyl thiab Hydroperoxyl Radicals: Kev Kawm Quantum Chemical thiab Computational Kinetics. J. Org. Chem. Xyoo 2012, 77, 3868-3877. [CrossRef] [PubMed]
27. Li, XC; Hu, QP; Jiang, SX; Li, F.; Lin, J.; Han, L.; Hong, YL; Lu, WB; Gao, YX; Chen, DF Flos Chrysanthemi Indici Tiv thaiv Hydroxyl-induced puas tsuaj rau DNA thiab MSCs ntawm Antioxidant Mechanism. J. Saudi Chaw. Soc. Xyoo 2015, 19, 454-460.
28. Amic, ib.; Markovic, Z.; Markovic, JMD; Stepanic, V.; Lucic, IB; Amic, D. Rau kev txhim kho kev kwv yees ntawm Dawb Radical Scavenging Potency ntawm Flavonoids: Qhov tseem ceeb ntawm Ob Chav PCET Mechanisms. Khoom noj khoom haus Chem. Xyoo 2014, 152, 578-585.
29. Lee, CH; Yoon, JY UV Direct Photolysis ntawm 2,2'-and-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) hauv Aqueous Solution: Kinetics thiab Mechanism. J. Phom. Photobiol. A 2008, 197, 232-238.
30. Aliaga, C.; Lissi, EA Reaction ntawm 2,2'-Azinobis(3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) Derived Radicals nrog Hydroperoxides. Kinetics thiab Mechanism. Int. J. Chem. Kinet. 1998, 30, { {8}}.
31. Osman, AM; Wong, KKY; Fernyhough, A. ABTS Radical-Driven Oxidation ntawm Polyphenols: Kev rho tawm thiab cov txheej txheem Elucidation ntawm Covalent Adducts. Biochem. Biophys. Res. Pawg. Xyoo 2006, 346, 321-329.
32. Osman, AM Multiple Pathways of the Reaction of 2, 2-Diphenyl-1-Picrylhydrazyl Radical (DPPHe) nrog (ntxiv )-catechin: Cov pov thawj rau Kev Tsim Cov Khoom Siv Covalent ntawm DPPHe thiab Daim Ntawv Oxidized ntawm polyphenol. Biochem. Biophys. Res. Pawg. Xyoo 2011, 412, 473-478.
33. Lopez-Munguia, A.; Hernandez-Romero, Y.; Pedraza-Chaverri, J.; Miranda-Molina, A.; Regla, ib.; Martinez, UA; Castillo, E. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity thiab Theoretical Study of their Free Radical Scavenger Mechanism. PLoS IB 2011, 6, e20115.
34. Perron, NR; Brumaghim, JL Kev Tshawb Fawb Txog Kev Tiv Thaiv Cov Tshuaj Tiv Thaiv Cov Txheej Txheem ntawm Polyphenol Cheebtsam cuam tshuam nrog hlau khi. Cell Biochem. Biophys. Xyoo 2009, 53, 75-100.
35. Devos, D.; Moreau, C.; Devedjian, JC; Kluza, J.; Perrault, M. Laloux, C.; Jonneaux, UA; Ryckewert, G.; Garcon, G.; Rouaix, N.; ua al. Targeting Chelatable Iron raws li kev kho tus qauv hauv Parkinson's Disease. Antioxidant. Redox teeb liab. Xyoo 2014, 21, 195-210.
36. Cekic, SD; Baskan, KS; Totem, E.; Apak, R. Hloov Kho Cupric Txo Antioxidant Capacity (CUPRAC) Kev Ntsuam Xyuas Kev Ntsuas Antioxidant Capacities ntawm Thiol-muaj Proteins hauv Admixture nrog Polyphenols. Talanta 2009, 79, 344-351.
37. Li, XC; Chen, NWS; Maiv, Y.; Wen, IB; Wang, XZ Concordance ntawm Antioxidant Kev Ua Haujlwm hauv vitro thiab Chemical Cheebtsam ntawm Radix Astragali (Huangqi). Nat. Prod. Res. Xyoo 2012, 26, 1050-1053.
38. Chen, DF; Li, XC; Xu, ZW; Liu, XB; Du, SH; Li, H.; Zhou, JH; Zeng, HP; Hua, ZC Hexadecanoic Acid los ntawm Buzhong Yiqi Decoction Induced Proliferation of Bone Marrow Mesenchymal Stem Cells. J. Med. Food 2010,13, 967-975.
39. Li, X.; Liu, JJ; Lin, J.; Wang, TT; Huang, YJ; Lin, YQ; Chen, DF Cov Kev Tiv Thaiv Kev Tiv Thaiv ntawm Dihydromyricetin tawm tsam » OH-Induced Mesenchymal Stem Cells Kev puas tsuaj thiab Mechanistic Chemistry. Molecules 2016, 21, 604.
40. Wang, GR; Li, XC; Zeng, HP Synthesis, Antioxidation kev ua ntawm (E)-9-p-Tolyl-3-2-(8-hydroxy-quinol- 2-yl)vinyl-carbazole thiab (E){{8 }}(p-Anisyl)-3-2-(8-hydroxy-quinol-2-yl)vinyl-carbazole thiab Lawv Induction Proliferation ntawm Mesenchymal Stem Cells. Acta Chim. Kev txhaum. Xyoo 2009, 67, 974-982.
41. Li, X.; Wei, G.; Wang, X.; Liu, D.; Deng, R.; Li, H.; Zhou, J.; Li, Y.; Zeng, H.; Chen, D. Targeting ntawm Shh Txoj Kev los ntawm atractylenolides txhawb kev sib txawv chondrogenic ntawm mesenchymal qia hlwb. Biol. Pharm. Bull. Xyoo 2012, 35, 1328-1335.
42. Li, XC; Gao, YX; Li, F.; Liang, AF; Xu, ZM; Bai, Y.; Maiv, WQ; Han, L.; Chen, DF Maclurin tiv thaiv hydroxyl radical-induced kev puas tsuaj rau mesenchymal qia hlwb: Antioxidant kev ntsuam xyuas thiab kev pom kev siv tshuab. Chem. Biol. Sib tham sib. Xyoo 2014, 219, 221-228.
Qauv Muaj: Ib qho piv txwv ntawm cov khoom sib xyaw polyumoside muaj los ntawm cov kws sau ntawv.
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