Ntu 2: Kev sib cuam tshuam ntawm Ascorbic Acid, 5-Caffeoylquinic Acid, Thiab Quercetin-3-Rutinoside nyob rau hauv qhov muaj thiab tsis muaj hlau thaum lub sij hawm ua cua sov thiab lub zog ntawm Antioxidant Activity

Yog xav paub ntxiv. hu rau:tina.xiang@wecistanche.com

Nyem qhov txuas mus kawm ntu 1 cov ntsiab lus:
https://www.xjcistanche.com/news/part1-interactions-of-ascorbic-acid-5-caffeo-54916714.html

3. Kev sib tham

3.1.Structure-Activity Relationship of Ascorbic Acid, 5-Caffeoylquinic Acid, thiab Quercetin-3-Rutinoside

Thawj qhov taw qhia ntawm AOA, ntsuas rau cov khoom sib txawv nyob ntawm tus qauv-kev sib raug zoo, tshwm sim los ntawm cov pab pawg ua haujlwm. Sib piv tag nrho peb cov tshuaj ntsuam xyuas, ascorbic acid muaj qis tshaj AOA, ua raws li 5-caffeoylquinic acid, thaumquercetin -3-rutinosidemus txog qhov siab tshaj AOA. Csepregi et al. [14] pom tib qhov kev txiav txim thaum muab piv rau AOA ntawm peb lub tebchaw. Qhov qeb duas no tuaj yeem piav qhia los ntawm tag nrho cov pawg hydroxy: quercetin-3-rutinoside muaj kaum, 5-caffeoylquinic acid muaj tsib, thiabascorbic acidmuaj plaub. Rauflavonoids, tag nrho cov nyiaj ntawm hydroxy pawg thiab lawv qhov cuam tshuam rau cov txheej txheem ntawm AOA tau qhia yav dhau los los ntawm Burda thiab Oleszek [15]. Hydroxy pawg muaj txiaj ntsig tshwj xeeb hauv cov qauv enediol, vim lawv tuaj yeem yooj yim oxidize rau diketones [8]. Nyob rau hauv cov tshuaj ntsuam xyuas cov qauv endiol kuj tseem ceeb heev rau lub peev xwm tsim complexes nrog hlau ions [16-18]. Cov qauv endiol tshwm sim hauv quercetin-3-rutinoside thiab 5-caffeoylquinic acid molecules nyob rau hauv lub nplhaib phenyl, uas kuj yuav cuam tshuam rau AOA muaj zog ntawm cov tebchaw. Cov kev tshawb fawb ntxiv pom tias AOA kuj tuaj yeem cuam tshuam los ntawm lwm cov qauv molecule. Phenolic acids tuaj yeem cuam tshuam los ntawm pawg carboxylic acid, piv txwv li, hydroxyphenyl acetic acid (R-CH=CH-COOH) yog ib pawg tsis muaj hluav taws xob rho tawm, piv rau hydroxycinnamic acid (R-CH,-COOH) xws li raws li caffeic acid nyob rau hauv 5-caffeoylquinic acid [19]. Hauv flavonoids, aglycones muaj AOA siab dua li cov sib txuas glycosides [20,21], nyob rau hauv cov ntaub ntawv ntawm quercetin -3-rutinoside, glycoside, AOA ntawm aglyconequercetinyog li ntawd siab dua [14]. Muaj ntau ntau pab pawg ua haujlwm sib txawv uas tuaj yeem cuam tshuam rau AOA thiab, yog li ntawd, nws yog ib qho tseem ceeb uas yuav tsum siv cov kev ntsuam xyuas sib txawv nrog cov txheej txheem sib txawv, xws li kev hloov hluav taws xob ib leeg (SET), hydrogen atom hloov (HAT), thiab sequential proton-tsis electron. Hloov pauv (SPLET) rau kev kuaj pom. Txhua lub tshuab thiab txawm tias siv cov tshuaj reagent tuaj yeem kuaj xyuas cov qauv sib txawv ntawm cov khoom siv bioactive [22].

3.2.Influence of Thermal processing and interaction of Structurally Different Antioxidants on the Antioxidant Activity in the absence of Mineral Hlau Hlau

AOA ruaj khov nyob rau lub sijhawm txuas ntxiv ntawm 40 min ntawm cov txheej txheem thermal qhia tias thermal degradation tsis tseem ceeb tshaj qhov pib xav. Tsis tas li ntawd, HPLC cov ntaub ntawv tau pom tias cov ntshiab 5-caffeoylquinic acid thiab quercetin-3-rutinoside tau ruaj khov, nrog rau qhov siab tshaj plaws degradation ntawm 20 feem pua, thaum lub sij hawm ua thermal. Cov kev tshawb fawb yav dhau los tau ua pov thawj qhov ruaj khov ntawm 5-caffeoylquinic acid mus txog qhov kub ntawm cov dej ib txwm [23], thaum Dawidowicz thiab Typek[24] pom cuaj qhov sib xyaw ua ke, tom qab cua sov 5-caffeoylquinic acid rau 5 h hauv qab reflux. Rau ascorbic acid concentration, degradation tom qab 40 min ntawm kev ua noj tau pom. Cov yam ntxwv cuam tshuam, ua rau ascorbic acid degradation yam tsis muaj hlau hauv cov kua dej, tuaj yeem yog pH qhov tseem ceeb, pom lub teeb, oxidation, qhov kub thiab txias, thiab qhov sib txawv [25,26]. Hauv qhov kev tshawb fawb no, qhov ntsuas kub thiab qhov concentration feem ntau yuav ua lub luag haujlwm tseem ceeb, vim tias cov txheej txheem oxidative raug txo kom tsawg vim qhov chaw roj tsawg hauv cov microtubes. Nws muaj peev xwm hais tias cov roj seem tseem txaus rau degradation nyob rau hauv aerobic tej yam kev mob. Cov xwm txheej sib txawv ua rau cov khoom sib txawv [27,28], yog li hauv txoj kev tshawb no, tom qab 40 min ntawm kev ua noj, cov khoom ntawm ob qho tib si tuaj yeem pom. Yuan thiab Chen [28] tau tshaj tawm tias furfural, 2-furoic acid, 3-hydroxy-2-pyrrone, thiab ib yam khoom tsis paub yog cov khoom degradation loj ntawm ascorbic acid hauv cov tshuaj aqueous nyob ntawm tus nqi pH . Shinoda et al. [29,30] pom hauv cov kua txiv kab ntxwv cov khoom degradation furfural, 2-furoic acid, 5-hydroxymaltol, 3-hydroxy-2-pyrrone, thiab 5-(hydroxymethyl ) cov plaub hau. Hsu et al. [31] txheeb xyuas ascorbic acid hauv cov tshuaj ethanolic thiab kuaj pom 2-furoic acid thiab 3-hydroxy-2-pyrrone. Nyob ntawm cov xwm txheej aerobic lossis anaerobic, cov tshuaj ascorbic acid derivatives (peaks 3 thiab 4) tej zaum yuav yog furfural, 2-furoic acid, los yog 3-hydroxy-2-pyrrone los yog intermediates. Cov ascorbic acid txo qis hauv qhov kev sib txuas ntawm koob tshuaj, thiab ntau dua ntawm ascorbic acid pom qhov txo qis qis dua thaum ua noj. Ascorbic acid zoo li stabilize nws tus kheej nyob rau hauv ntau dua, xav tias yog vim hydrogen bonds thiab van de Waals zog [32].

Hauv kev sib xyaw binary thiab ternary, feem ntau ntxiv cov teebmeem ntawm AOA tau pom. Hauv kev sib xyaw binary, yog tias cov khoom siv nrog AOA siab dua hauv nws cov concentration, AOA ntawm lawv cov kev sib xyaw kuj nce ntxiv. Lwm cov kev tshawb fawb kuj pom muaj qhov cuam tshuam ntxiv ntawm (ntxiv rau )-catechin (200 um) nrog ascorbic acid (50-200 mg/L)[33], thiab ntawm binary sib xyaw ntawm cov monoterpenes sib txawv[34]. Tsuas yog nyob rau hauv qhov kev ntsuam xyuas DPPH, cov teebmeem antagonistic tau pom nyob rau hauv 5-caffeoylquinic acid ua ke nrog quercetin-3-rutinoside thiab nyob rau hauv ternary mixs nrog doubled quercetin-3-rutinoside concentrations. Qhov no tuaj yeem tshwm sim los ntawm kev taw qhia ntawm cov molecules nyob rau hauv qhov chaw, tshwj xeeb tshaj yog quercetin -3-rutinoside, raws li ib tug loj molecule, thiab steric nkag tau ntawm DPPH radical molecule [35]. Hauv kev soj ntsuam TPC, kev sib xyaw ntawm ascorbic acid thiab 5-caffeoylquinic acid ua rau muaj zog antagonistic los ntawm 1: 2 piv, thaum rov qab sib tov nrog 2: 1 piv ua rau muaj kev sib koom ua ke muaj zog, tshaj qhov sib npaug ntawm cov raws li AOAs. Ob chav concentration ascorbic acid tuaj yeem muab, nyob rau hauv cov xwm txheej no, kev txhawb nqa ntxiv rau AOA, vim tias nws tus kheej ruaj khov ua raws li kev ruaj ntseg ntawm lwm cov molecules, ua qauv qhia los ntawm 5-caffeoylquinic acid hauv qhov kev sim no. Hauv cov kua txiv pomegranate-nectarine, nruab nrab ntawm ntuj phenols thiab ascorbic acid, tib qhov kev sib cuam tshuam tau pom, thaum nyob rau hauv cov kua txiv hmab txiv ntoo ua rau muaj kev cuam tshuam los ntawm kev nce ascorbic acid concentration tau pom [36]. Cov txiaj ntsig no qhia tias kev sib xyaw ntawm ascorbic acid, 5-caffeoylquinic, thiab quercetin-3-rutinoside ua tiav AOA lub peev xwm siab tshaj plaws thaum lub zog tshaj plawsantioxidantsmuaj abundantly.

Nyem kom paub ntau ntxiv txog cov khoom

3.3. Kev cuam tshuam ntawm Mineral Hlau

The addition of iron to the samples had the most influence on changes in their AOA. Contrary to the hypothesis, based on the pro-oxidative activity of iron, the AOA increased, compared to the same substance or substance mixture without iron. Iron may act like a catalyst itself or forms metal chelates, which are effective catalysts. The changed stoichiometry of the chelates can form additional radical-scavenging metal centers [18], which explains the increased AOA. Further tests showed that reduced ferrous iron (50-100%,v/o)itself interacts with the TEAC(0.266-0.538 mol TE/mol iron), DPPH(0.210-0.495 mol TE/mol iron), and TPC(16.65-31.82g GAE/mol iron) assay reagents, while oxidized ferric iron does not (data not shown). In the presence of iron, 5-caffeoylquinic acid had the highest AOA, followed by quercetin-3-rutinoside and ascorbic acid in TEAC and DPPH assays. This may be due to the changed stoichiometry by metal chelation. The AOA ranking detected by the TPCassay stayed the same, as in the absence of iron: quercetin-3-rutinoside >5-caffeoylquinic acid>ascorbic acid. Txawm li cas los xij, qhov sib ntxiv ntawm cov hlau rau cov khoom sib xyaw ua rau muaj kev cuam tshuam rau TEAC thiab DPPH kev soj ntsuam cov txiaj ntsig, thaum TPC kev ntsuam xyuas tsis muaj kev cuam tshuam ntau. Vim li no, nws yog ib qho tseem ceeb uas yuav tsum tau siv cov kev ntsuam xyuas sib txawv nrog cov txheej txheem ua haujlwm sib txawv thaum ua haujlwm nrog cov qauv hlau nplua nuj.

Tsuas yog hauv DPPH kev soj ntsuam AOA ntawm ntshiab quercetin-3-rutinoside, thiab ua ke nrog ascorbic acid, equimolar lossis non-equimolar nrog ob npaug ntawm quercetin -3-rutinoside concentrations, puas muaj qis dua qhov muaj hlau. Boligon et al. [351 piav qhia tias DPPH kev soj ntsuam kuaj pom cov tshuaj tua kab mob me me zoo dua, vim tias muaj kev nkag mus tau zoo ntawm cov radicals. Piv txwv li, quercetin-3-rutinoside nrog ob txoj hlua khi tuaj yeem tsim cov complexes loj heev nrog hlau, thiab yog li tsis tuaj yeem nkag mus rau qhov kev ntsuam xyuas. Raws li tau hais los ntawm Kejic et al. [8], qhov no yuav piav qhia los ntawm kev tsim cov supramolecular complexes ntawm kev sib koom tes ntawm cov hlau ions. Nyob rau hauv kev sib xyaw nrog ascorbic acid, uas muaj peev xwm tsim cov mix-valence complexes nyob rau hauv ib tug 1: 2 ratio [12], tej zaum, cov loj sib tov complexes yuav tsis cuam tshuam nrog DPPH radical. Contrary to the results in this study, other studies [18,37,38] pom, by using the DPPH assay, that quercetin{13}}rutinoside complexes are more effective antioxidants than pure quercetin-3-rutinoside. Nws paub tias chelate complexes ntawm flavonoids thiab hlau ions tuaj yeem cuam tshuam cov kev ua haujlwm radical ntawm complexed hlau ions [39]. Qhov sib txawv tsuas yog tias hauv txoj kev tshawb no ammonium hlau (II) sulfate tau siv, tsis yog hlau (ⅡI) chloride lossis sulfate, siv hauv kev ua haujlwm ntawm Symonowics thiab Kolandek [39].

Synergistic teebmeem ntawm ascorbic acid thiab 5-caffeoylquinic acid nyob rau hauv lub xub ntiag ntawm hlau nyob rau hauv lub 2: 1 piv, thiab antagonistic teebmeem nyob rau hauv 1: 2 piv, tau pom. Kev sib xyaw ntawm 5-caffeoylquinic acid nrog hlau tsis pom zoo hauv vivo [6]. Txawm li cas los xij, txoj kev tshawb fawb no tau pom tias, hauv vitro, ob npaug ntawm ascorbic acid piv rau 5-caffeoylquinic acid tuaj yeem muaj kev sib koom ua ke ntawm AOA. Kev tshawb nrhiav ntxiv ntawm qhov sib piv tuaj yeem pom tias cov txiaj ntsig zoo tuaj yeem ua tiav hauv vivo.

Raws li HPLC cov ntaub ntawv, ntxiv hlau rau cov qauv yuav muaj qhov tsawg kawg nkaus ntawm nws cov catalytic kev ua si, raws li tsuas yog cov ascorbic acid concentration nyob rau hauv 0 min cov qauv siav txo. Hauv cov qauv ascorbic acid siav, tsis zoo li cov qauv uas tsis muaj hlau, ntau dua ascorbic acid concentrations coj mus rau qhov sib piv ntawm cov degradation ntau dua.5-Caffeovlquinic acid thiab quercetin-3-rutinoside xav tau qhov kub thiab sijhawm ntxiv, nrog rau Kev sib cuam tshuam hauv kev sib tov, rau kev ua haujlwm catalytic ntawm hlau ua haujlwm. Quercetin-3-rutinoside, nyob rau hauv lub xub ntiag ntawm hlau, tsuas yog txo nyob rau hauv binary sib tov nrog ascorbic acid tom qab thermal ua. Qhov no tuaj yeem yog vim qhov tseeb tias flavonoid ua haujlwm tseem ceeb antioxidant thiab tom qab ntawd cov khoom sib xyaw radical reacts nrog ascorbic acid, regenerating thawj compound [18]. 5-Caffeoylquinic acid zoo li tshwj xeeb, vim nws tiv thaiv quercetin-3-rutinoside, thaum ascorbic acid tsis tuaj yeem tiv thaiv quercetin-3-rutinoside. Li no, 5-caffeoylquinic acid tej zaum yuav yog lub ntsiab molecule rau stabilizing lub system nyob rau hauv ua ke nrog nrog hlau thiab thermal ua. Qhov no hypothesis ntawm 5-caffeoylquinic acid raws li ib tug stabilizing molecule tau paub ntxiv nyob rau hauv lub ternary mixs. Ntawm no, quercetin-3-rutinoside yeej ib txwm nyob ruaj khov los ntawm 5-caffeoylquinic acid, yog li ntawd txawm tias muaj ascorbic acid tsis txo qis hauv quercetin-3-rutinoside concentration tau pom. Hauv lwm txoj kev tshawb fawb, 5-caffeoylquinic acid tau qhia txog kev tiv thaiv cov khoom tiv thaiv kev degradation ntawm anthocyanins los ntawm cov txheej txheem co-pigmentation [40]. Cov khoom lag luam degradation tshiab, muaj peev xwm siab dua AOA, tshwm sim los ntawm tag nrho peb cov khoom nyob rau hauv muaj hlau. Caffeic acid (peak 6) tau txheeb xyuas tias yog 5-caffeoylquinic acid rhuav tshem cov khoom (cov ntaub ntawv tsis qhia). Qhov no coj mus rau qhov kev xav tias lwm yam khoom yuav yog quinic acid los yog ib qho ntawm cuaj cov khoom siv tau piav qhia los ntawm Dawidowicz thiab Typek [24]: quinic acid;(1S,3R,4R,5R)-5-[{{24] }}(3,4-dihydroxyphenyl)-2-hydroxypropanoyl]-1,4,5-trihydroxy-cyclohexane carboxylic acid;(1S3R,4R,5R){36}} [3-(3,4-dihydroxyphenyl)-3-hydroxypropanoyl]-1,4,5 trihydroxycyclohex-anecarboxylic acid; trans 3-O-caffeoylquinic acid; trans 5-O-caffeoylquinic acid; trans 4-O-caffeoylquinic acid; caffeic acid; cis-5-O-caffeoylquinic acid; 4, 5-dicaffeoylquinic acid. Rau quercetin-3-rutinoside, cov khoom degradation tshwm nyob rau hauv chromatogram, uas tsis tuaj yeem txheeb xyuas.

3.4. Muaj peev xwm tsim Chelates nrog Ferric (Fe3 ntxiv) thiab Ferrous Iron (Fe2 ntxiv)

Nyob rau hauv tag nrho cov qauv uas muaj ascorbic acid, ferric hlau raug txo kom ferrous hlau. Hauv cov txheej txheem no,ascorbic acidsiv lub tshuab hluav taws xob los ntawm ferric hlau thiab txo nws mus rau ferrous hlau, thiab dhau los ua radical nws tus kheej. Cov tsis ruaj tsis khov radical converts sai heev rau dehydroascorbic acid thiab ntxiv degradation khoom [12]. Vim tias tsis muaj hydroxy peroxide, cov tshuaj tiv thaiv rov qab rau ferric hlau ntawm Fenton lub voj voog tsis tuaj yeem ua tau. Hauv 0 min siav binary sib tov, ntau tshaj 20 feem pua ​​​​ntawm cov hlau tau khi rau hauv lub xub ntiag ntawm ascorbic acid. Nws paub tias ascorbic acid tsim complexes nrog hom hlau thiab lwm yam hlau ions los ntawm chelation ntawm 3-O thiab 2-O nuclei tom qab hydrogen hloov chaw los ntawm 3-OH thiab 2- OH pawg [16,17,41. Nws tuaj yeem tsim cov khoom sib xyaw-valence hlau-ascorbate complexes [42]. Txawm li cas los xij, ascorbic acid yog tus neeg saib xyuas tsis muaj zog thiab, tom qab ua noj, tsuas yog cov kab ntawm cov hlau khi tau pom hauv cov qauv ntshiab thiab sib xyaw thaum muaj ascorbic acid. Tsis tas li ntawd, vim cov txheej txheem ua noj, ascorbic acid tawg mus rau cov khoom degradation, uas zoo li tsis tuaj yeem chelate nrog hlau.

5-Caffeoylquinic acid yog ib qho tsis zoo reductant. Ferric hlau raug txo los ntawm 5-caffeoylquinic acid, thiab nrog kev ua noj ua haus ntev, txo qhov nce ntxiv.5-Caffeoylquinic acid chelates nrog ferric hlau nyob rau hauv ib lub ligand rau hlau hloov pauv [17].5-Caffeoylquinic acid nqa ib sab ua tau khi ntawm 3,4 endiol qauv ntawm caffeic acid. Qhov no tuaj yeem yog qhov qhia tau tias yog vim li cas caffeic acid yog bioactive ib feem ntawm 5-caffeoylquinic acid, thaum quinic acid yuav luag tsis muaj AOA [43]. Endiols inhibited OH tsim vim tsim cov hlau complex [41] nyob rau hauv ib tug-rau-ib piv [44,45]. Lamy et al.[46] xaus lus tias 5-caffeoylquinic acid tsim monomeric complexes, whereas Kiss li al. [47] pom hom oligomeric. Contrary to yav dhau los kev tshawb fawb [17,48], tsuas yog ib tug kab ntawm khi hlau tau pom nyob rau hauv lub xub ntiag ntawm 5-caffeoylquinic acid ua ntej thiab tom qab thermal ua. Qhov no tuaj yeem piav qhia los ntawm qhov nruab nrab pH nyob rau hauv txoj kev tshawb no, thaum lwm cov kev tshawb fawb ua haujlwm hauv cov kua qaub nruab nrab, raws li tus nqi pH ntawm 1-2.5 hauv tib neeg lub plab [48]. Cov dej nag dub tau pom nyob rau hauv cov qauv khaws cia tom qab ob peb teev, uas yog qhov qhia txog ntawm 5-caffeoylicquinic acid-ferric hlau complexes. Tsis ntev los no cov qauv sib xyaw tau siv rau kev tsom xam, yog li tsim cov complexes ntawm qhov nruab nrab pH yuav tsum tau siv sijhawm ntev dua. Hlau complexes nrog caffeic acid pom me ntsis scavenging kev ua si [49]. Tsis tas li ntawd, tsis muaj pov thawj spectrophotometric rau cov tshuaj tiv thaiv ntawm quinic acid thiab ferric hlau [48]. Contrary to 5-caffeoylquinic acid, nyob rau hauv lub quercetin-3-rutinoside qauv, khi hlau tau pom, txawm tias tom qab thermal ua.

Quercetin-3-rutinoside txo ferric hlau rau ferrous hlau nrog qhov tsawg kawg nkaus nce los ntawm lub sijhawm ua noj ntev. Qhov no txo ​​qis kev ua haujlwm ntawm quercetin-3-rutinoside yav dhau los tau piav qhia los ntawm Mira et al. [50]. Lub ferric txo kev ua haujlwm ntawm quercetin-3-rutinoside tau kuaj pom hauv 3-rutinoside, 5,7,3',4'-OH [50]. Kev sib cuam tshuam nruab nrab nrog ferric hlau tuaj yeem piav qhia los ntawm tus lej qis ntawm -OH pawg, uas ua rau qis qis qis qis ntawm chelation sab [50]. Flavonoids tuaj yeem chelate nrog hlau ions ntawm peb qhov kev sib koom ua ke: (i) nruab nrab ntawm 5-hydroxy thiab 4-carbonyl pawg, (ii) nruab nrab ntawm 3-hydroxy thiab 4-carbonyl pawg, thiab (ii) nruab nrab ntawm 3', A'-hydroxy pawg hauv Nqa [39]. Quercetin-3-rutinoside siv cov hlua khi (i) thiab (i)[9], thiab ntawm 3- pawg hydroxy, rutinoside txuas nrog. Spectral cov ntaub ntawv txawm pom tias hlau ions tsuas yog khi rau 3', A'-hydroxy pawg [18]. Chelates tau zoo dua nrog hlau hauv nws daim ntawv bivalent [50]. Hauv kev sib xyaw binary, quercetin-3-rutinoside tsis tuaj yeem tsim cov complexes thaum muaj ascorbic acid, tsis sib xws hauv ternary mixs. Yog tias muaj 5- caffeoylquinic acid, muaj cov hlau khi me me. Qhov no qhia tau tias 5-caffeoylquinic tiv thaiv quercetin-3-rutinoside molecules nyob rau hauv lub xub ntiag ntawm ascorbic acid, yog li quercetin-3-rutinoside tuaj yeem tsim cov complexes nrog hlau.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1.Chemical

ABTS(2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt)(≥98%)was obtained from Sigma-Aldrich (Steinheim, Germany), DPPH*(2,2-diphenyl-1-picrylhydrazyl) radical (95%) and Trolox(97%)were obtained from Thermo Fisher (Kandel, Germany). Folin-Ciocalteu phenol reagent was purchased from Merck(Darmstadt, Germany). HPLC grade methanol, acetonitrile(HPLC grade), glacial acetic acid (100%, p.a.), sodium acetate trihydrate (≥99.5% p.a.), potassium thiocyanate (≥98.5%, p.a., ACS),2,2'-dipyridyl (≥95%), hydrochloric acid (≥25%, p.a., ISO), gallic acid monohydrate (≥99%), potassium persulfate (≥99%), rutin trihydrate (working standard), chlorogenic acid (working standard), L-(+)-ascorbic acid (working standard), sodium carbonate(>99 feem pua), ferric ammonium sulfate dodecahydrate, thiab ferrous ammonium sulfate hexahydrate tau yuav los ntawm Carl Roth (Karlsruhe, Lub teb chaws Yelemees).

4.2.Sample

Aqueous solutions thiab mixs of authentic standard of ascorbic acid, {{{0}}}caffeoylquinic acid, thiab quercetin-3-rutinoside tau npaj ua cov tshuaj dawb huv thiab sib tov sib txawv. Txhua qhov sib xyaw ua ke binary tau ua nyob rau hauv equimolar ratios, thiab non-equimolar ratios nrog ib tug compound doubled. Ternary mixs kuj tau npaj nyob rau hauv equimolar ratios, raws li zoo raws li nyob rau hauv non-equimolar ratios nrog ib los yog ob lub tebchaw doubled, feem. Cov tshuaj tua kab mob antioxidants kawg yog 0.3 mM rau txhua qhov kev sim tshuaj. Tsis tas li ntawd, txhua qhov kev sim tau rov ua dua tom qab qhov sib ntxiv ntawm 0.3 mM hlau ({{10}}.15 mM ferrous hlau thiab 0.15 mM ferric hlau) nyob rau hauv tag nrho ib qho sib tov. Kev xaiv cov tshuaj antioxidants thiab hlau tsis yog raws li lub cev lossis qib zaub mov, lawv yog raws li molar pawg los kawm txog kev cuam tshuam ntawm molecular. Yog li ntawd, ib qho piv txwv ntawm tag nrho cov antioxidants thiab hlau tau tsim. Tag nrho cov khoom xyaw tau siav rau 0, 10, 20, thiab 40 min hauv dej kub npau npau, thiab tom qab ntawd txias rau hauv dej khov kom tsis txhob muaj cua sov. Qhov no tau ua tiav rau peb qhov kev hloov pauv ywj pheej.

4.3.Photometric Measurements

Antioxidants(ntshiab lossis sib xyaw) thaum tsis muaj thiab muaj hlau raug ntsuas rau lawv qhov kev txo qis tag nrho thiab kev ua haujlwm antioxidant hauv peb qhov kev ywj pheej kev rov ua dua uas siv cov txheej txheem high-throughput hauv 96-cov phiaj zoo (SynergyTM HTX Multi-Mode Microplate Reader, BioTek Instruments, Winooski, VT, USA). Cov kev ntsuam xyuas sib txawv tau siv, vim muaj cov tshuaj tiv thaiv sib txawv: kev hloov hluav taws xob ib leeg (SET) thiab hydrogen atom hloov pauv (HAT). Thaum qhov kev ntsuam xyuas TEAC thiab TPC yog raws li SET [17,18], rau qhov kev xeem DPPH, cov ntaub ntawv tsis paub meej yog tias nws yog raws li SET, HAT, lossis txawm tias kev sib xyaw ntawm ob lub tshuab no [{{6} }]. Ib txoj kev tshawb fawb tsis ntev los no los ntawm Foti [51] pom tias phenols tuaj yeem hnov ​​​​mob nrog DPPH los ntawm kev hloov pauv ntawm proton-lossis electron (SPLET), ua ke ntawm ob lub tswv yim. Cov yam ntxwv, xws li nruab nrab polarity thiab ionization muaj peev xwm, cuam tshuam rau cov txheej txheem tseem ceeb.

4.3.1. Tag nrho cov ntsiab lus Phenolic (TPC)

Tag nrho cov ntsiab lus phenol (TPC) tau txiav txim siab siv txoj kev Folin-Ciocalteu hauv 96-cov phaj zoo, tau piav qhia yav dhau los los ntawm Bobo-Garcia li al.[52] nrog qee qhov kev hloov kho.

Briefly, 10 μL Folin-Ciocalteu reagents were mixed with a 50 μL sample, and afterward 100 μL Na, CO3 was added. The 96-well plate was incubated at 37 °C（±0.2°C） and with constant orbital shaking at a moderate speed (237 CPM, 4 mm) for 14 min. After a 1 min resting period, the absorbance was measured at 736 nm. Results were expressed as gallic acid equivalents (mg GAE/ mol Antioxidant), using a standard curve ranging from 5.97 to 59.7 ug gallic acid/mL(R~>0.99). Lub npe nrov ntawm qhov kev sim no yog kev dag ntxias vim tias Folin-Ciocalteu reagent kuj ua rau cov tsis-phenolics, xws li cov vitamins thiab minerals [22]. Nws piav qhia tau zoo dua "tag nrho cov haujlwm txo qis" ntawm bioactive compounds.

4.3.2. Trolox Equivalent Antioxidant Capacity (TEAC)

Cov tshuaj tiv thaiv antioxidant tau txiav txim siab siv TEAC kev soj ntsuam hauv 96- phaj zoo nrog qee qhov kev hloov kho. Ib qho kev daws teeb meem nrog 9.6 mg ABTS thiab 1.66 mg poov tshuaj persulfate tau ntim nrog H2O rau 25 mL tau npaj thiab muab tso rau hauv qhov tsaus ntuj ntawm chav tsev kub rau 12-16 h. Los ntawm cov kev daws teeb meem no, TEAC ua hauj lwm kev daws teeb meem, uas muaj 5mL Tshuag cov tshuaj tau sau txog 25 mL nrog 100 feem pua ​​MeOH, tau npaj.

Briefly, 10 μL of the sample was mixed with 150 μL of the TEAC working solution. After a 5 min incubation, the plate was shaken orbitally at a moderate speed for 1 min, followed by a 1 min resting period. The absorbance was measured at a wavelength of 734 nm. The TEAC was expressed as Trolox equivalents (mol TE/mol Antioxidant), using a standard curve ranging from 0.025-0.8 mM Trolox(R->0.98).

4.3.3. DPPH * Radical Scavenging

Covantioxidantactivity was determined using the modified DPPHmethod for 96-well plates. A DPPHworking solution with 7.88 mg DPPH filled up to 100 mL was prepared. Briefly, 20 μL of the sample was mixed with 180 ul of the DPPH working solution and incubated in the dark for 28 min at room temperature. After 1 min orbital shaking at a moderate speed and a 1 min resting time, the absorbance was measured at a wavelength of 515 nm. Results were expressed as Trolox equivalents (mol TE/mol Antioxidant), using a standard curve ranging from 0.025-0.8 mM Trolox(R2>0.98).

4.4. Synergism thiab antagonism

Rau kev tsom xam ntawm synergistic thiab antagonistic los ntawm kev ua haujlwm antioxidant, kev sib piv ntawm cov txiaj ntsig tau txais kev sim nrog cov txiaj ntsig theoretical suav los ntawm cov txiaj ntsig ntawm cov khoom ntawm ib tus neeg ntawm qhov sib xyaw ua ke tau ua [53]. Synergism piav qhia txog kev sib cuam tshuam ntawm ob lossis ntau yam khoom kom qhov sib xyaw ua ke ntau dua li qhov sib npaug ntawm txhua qhov kev ua yeeb yam sib cais. Contrary to this, antagonism yog ib qho tshwm sim uas qhov kev sib cuam tshuam ntawm ob lossis ntau yam khoom hauv kev sib xyaw ua ke muaj qhov cuam tshuam tag nrho uas tsawg dua qhov sib npaug ntawm lawv tus kheej cov teebmeem.

4.5.Kev txiav txim siab ntawm Ionic Hlau

The colorimetric determination of ferrous and ferric iron was modified according to Niedzielski et al. 【54】 for 96-well plates. Briefly, for ferrous iron detection, 20 μL acetate buffer(90 g sodium acetate trihydrate and 48 g acetic acid glacial filled up to 200 mL)and 20 μL 2,2'dipyridyl （0.5%, m/m） and, for ferric iron detection, 20 μL hydrochloric acid （2 M） and 20 μL potassium thiocyanate （5%, m/m）were pipetted into a 200 μL sample in the 96-well plate, incubated there for 10 min at room temperature, and the absorbance was measured for ferrous iron at520nm and for ferric iron at 470nm. Results were expressed in mM ionic iron/mM total iron, using a standard curve ranging for ferrous iron from 0.024-0.214mM(R'>0.99) and for ferric iron from0.005-0.178 mM(R->0.99). Qhov sib txawv ntawm tag nrho cov hlau thiab ionic hlau, qhov sib npaug ntawm ferrous thiab ferric hlau, yog cov hlau khi.

4.6.HPLC-DAD

Txhawm rau kom muaj nuj nqis ntawm cov tshuaj antioxidants ascorbic acid, 5-caffeoylquinic acid, thiab quercetin-3-rutinoside thiab degradation cov khoom muaj nyob rau hauv tib cov extracts siv rau photometric

measurements, a Shimadzu Prominence 20 high-performance liquid chromatography (HPLC) system equipped with a refrigerated SIL-20AC HT autosampler, CTO-10AS VP column oven, DGU-20A5 degasser, LC-20 AT liquid chromatograph quaternary pump, and an SPD-M20A diode array detector (DAD) was used. As a column for separation, a Supelco Ascentis⑧Express an F5 column (150× 3.0 mm, 5 um)equipped with a Supelco Guard column （5×3.0 mm, 5 μm） and a 0.2 micron SST Frit for UltraLite was used. The column temperature was set to 30°C. UV detection was at 245 nm for ascorbic acid, 320 nm for 5-caffeoylquinic, and 360 nm for quercetin-3-rutinoside. The mobile phase consisted of Eluent A (1% acetic acid (v/o), pH 2.5) and Eluent B(100% ACN). The separation was achieved using the following gradient program: 0-2.5 min.5% B:2.5-15 min.5-20%B:15-20 min,20%B;20-22.5 min, 20-5%B;22.5-30 min,5%B.The flow rate was 0.3 mL/min, and the sample injection volume was 30 μL. Standard calibration curves for the three substances 5-caffeoylquinic （0.5-0.15 μM; R2>0.99）, ascorbic acid （0.35-0.025 uM; R2>0.99）, and quercetin-3-rutinoside（0.35-0.025 μM; R2>0.99) tau npaj. Derived compounds tau tentatively txheeb xyuas los ntawm kev tshuaj xyuas cov qauv ntshiab nyob rau hauv lub xub ntiag thiab tsis muaj hlau ua ntej thiab tom qab thermal ua. Yog li ntawd, lub ncov tshiab yuav tsum tau muab los ntawm cov txheej txheem ntxig. Tsis tas li ntawd, cov kev xaiv sib xyaw tau raug ntsuas los ntawm HPLC-MS txhawm rau txheeb xyuas cov qauv tsim.

4.7. Kev txheeb cais

Microsoft Excel 2016 (Microsoft, Redmond, USA) thiab R Statistics (version 3.6.3, Hold-ing the Windsock, 2020) tau siv rau kev sim biostatistics thiab nthuav qhia thiab kos cov ntaub ntawv tau txais. Inferential txheeb cais rau kev ntsuam xyuas thiab txuas cov kev kho mob tau ua los ntawm kev siv peb txoj kev tsom xam ntawm qhov sib txawv (ANOVA), tom qab hoc Tukey's HSD test, thiab Pearson correlation. Cov pob R siv yog ggplot2 [55], txhais tau tias [56], thiab multcomp [57].

5. Cov lus xaus

Raws li cov lus piav qhia saum toj no, AOA ntawm ascorbic acid, 5-caffeoylquinic acid, thiab quercetin-3-rutinoside tau cuam tshuam los ntawm lawv cov qauv molecule, concentration, piv, thiab kev cuam tshuam nrog lwm cov antioxidants thiab hlau. Kev sib cuam tshuam tshwj xeeb zoo li ua lub luag haujlwm hauv AOA thaum sib txuas ascorbic acid thiab 5-caffeoylquinic acid. Ntawm no, muaj kev sib koom ua ke thiab antagonistic cuam tshuam. Qhov ntsuas kub muaj qhov cuam tshuam tsawg kawg nkaus ntawm AOA, thaum tib lub sijhawm ntsuas kub cuam tshuam rau kev ruaj ntseg ntawm txhua yam antioxidants hauv qee qhov sib xyaw, tshwj xeeb tshaj yog muaj hlau. Tsuas yog cov ascorbic acid concentration txo qis thaum tsis muaj hlau nrog lub sijhawm ua noj ntev thiab 5-caffeoylquinic acid concentration txo qis tsuas yog thaum muaj hlau, thaum quercetin-3-rutinoside concentration poob tsuas yog ua ke nrog ascorbic acid nyob rau hauv muaj hlau. Ua ke nrog cov hlau, 5-caffeoylquinic acid tuaj yeem tiv thaiv lwm cov molecules los ntawm kev txo qis hauv lawv cov concentration los ntawm kev ua kom sov.

Hauv cov nroj tsuag, kev sib xyaw ntawm ascorbic acid, 5-caffeoylquinic acid, thiab quercetin-3-rutinoside tsis yog tsuas yog ua tau tab sis muaj ntau. Li no, cov txiaj ntsig no muab cov kev paub yooj yim ntawm cov txheej txheem uas tshwm sim thaum ua noj zaub. Khoom noj khoom haus matrices yog complex thiab muaj suav tsis txheeb bioactive compounds, nrog rau enzymes, lwm yam minerals, los yog acids, uas hloov cov tshuaj tiv thaiv tej yam kev mob los yog reactants lawv tus kheej. Cov kev sib cuam tshuam nyuaj no nyob deb dhau ntawm qhov kev tshawb fawb no thiab cov txiaj ntsig zoo thiab kev sib cuam tshuam ntawmantioxidantsnyob rau hauv cov zaub siav yuav tsum tau hais txog yav tom ntej.

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