NMR-Based Metabolomics Profiling Rau Radical Scavenging Thiab Anti-Aging Properties Part 2

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

2.5. Kev faib tawm ntawm Tshuaj ntsuab Extracts los ntawm Thawj Tswj Hwm Kev Ntsuam Xyuas

Cov metabolite variation ntawm nplooj ntawm cov tshuaj ntsuam xyuas tau soj ntsuam ntxiv siv multivariate data analysis (MVDA). Tus Thawj Saib Xyuas Kev Tshawb Fawb (PCA), tus qauv lees paub, yog ib qho tsis muaj kev saib xyuas MVDA uas muab kev nkag siab loj ntawm kev sib koom ua ke ntawm cov qauv. Cov phiaj xwm PCA tau pom tias muaj kev sib cais ntawm cov tshuaj ntsuab rau hauv pawg, qhov chaw thauj khoom tau qhia txog cov metabolites uas muab kev sib cais [85,86]. Tus qauv PCA nthuav tawm lub cev muaj zog zoo (R2X=0.997) thiab qhov ntsuas tau siab (Q2=0.993) qhov twg qhov sib txawv ntawm R2X(cum) thiab Q2(cum) tsawg dua 0 .3, yog li qhia tau hais tias txhua yam ntawm cov tshuaj ntsuab extracts sib npaug thiab sib npaug pab rau kev soj ntsuam pab pawg sib cais. Qhov kev soj ntsuam no tau ua raws li Wheelock li al. [87].

Thov nias ntawm no kom paub ntxiv

Cov ntsiab lus tseem ceeb ntawm kev txheeb xyuas cov qhab nia tau pom tau tias cov tshuaj ntsuab tau xaiv tau muab cais ua ob pawg yam tsis muaj qhov zoo tshaj plaws raws li qhia hauv daim duab 4A. Tus Thawj Coj Cheeb Tsam (PC) 1 tau nthuav tawm cov qauv kev hloov pauv loj tshaj plaws, tom qab ntawd los ntawm PC2. PC1 thiab PC2 pab txhawb rau qhov feem pua ​​​​ntawm qhov sib txawv ntawm 29.7 feem pua ​​thiab 24.9 feem pua, raws li. Yog li, tag nrho qhov sib txawv ntawm kwv yees li 54.6 feem pua ​​​​tau piav qhia los ntawm cov PCs no. Cov txiaj ntsig tau los ntawm cov kab ke thauj khoom tau nthuav tawm cov metabolites lav rau kev sib cais ntawm cov tshuaj ntsuab mus rau qhov zoo ntawm PC1 (V.negundo thiab C.longa) thiab sab tsis zoo ntawm PC1 (P. minus, P. indica, O.javanica thiab C. caudatus)(Daim duab 4B).

Cov ntaub ntawv los ntawm kev thauj khoom kab ke ntawm PC1 pom tias cov phenolic tebchaw, feem ntau yog pawg flavonoid thiab phenolic acids, tau lav ris rau kev sib cais ntawm cov tshuaj ntsuab xaiv. Quercetin (1), quercetin-3-O-rhamnoside (2), quercetin-3-O-glucoside (3), quercetin-3-O-glucuronide (4), quercetin-3- O-arabinofuranoside (5), rutin (6), mvricetin derivatives (7), catechin (8), epicatechin (9), isorhamnetin (10), astragalin (11), chlorogenic acid (12), gallic acid (13), coumaric acid (14), ascorbic acid (15), formic acid (21), fumaric acid (22), 3-methylxanthine (26) thiab apigenin (28) cov ntsiab lus feem ntau siab dua hauv P. minus, P. indica , C. caudatus thiab O.javanica raws li lawv tau nyob rau hauv qhov tsis zoo ntawm cov phiaj xwm. Nyob rau hauv sib piv, V.negundo thiab C.longa tau txawv los ntawm lwm yam tshuaj ntsuab los ntawm muaj serotonin (27) thiab D-limonene (29) nyob rau hauv lawv cov extracts.

2.6. Kev sib raug zoo ntawm Bioactioities thiab Metabolite Siv Ib Feem Tsawg-Squares Analysis (PLS)

Txhawm rau nkag siab txog kev sib koom ua ke ntawm kev ntsuas bioactivities thiab metabolites pom nyob rau hauv cov tshuaj tshuaj ntsuam xyuas, PLS, raws li kev saib xyuas MVDA txheej txheem, tau siv los txheeb xyuas cov ntaub ntawv ywj pheej hloov pauv (NMR tshuaj hloov pauv ntawm cov metabolites) rau cov ntaub ntawv ntawm cov hloov pauv, uas yog qhov inhibition ntawm DPPH, ABTS thiab ORAC kev soj ntsuam, nrog rau kev tiv thaiv elastase thiab kev tiv thaiv-collagenase. Cov txheej txheem no tau siv vim tias PLS muaj qhov ua tiav zoo txhawm rau txuas cov kev sim bioactivities nrog cov metabolites, yog li tuaj yeem muab tus qauv rau kev kwv yees [88]. Los ntawm PLS tsom xam, kev sib raug zoo ntawm bioactivities xws li radical scavenging kev ua ub no thiab anti-aging zog nrog metabolites nyob rau hauv cov qauv yuav tsim. Yog li, cov metabolites uas yog lub luag haujlwm raws li cov cim bioactive tuaj yeem raug qhia.

Lub biplot yog qhov sib xyaw ntawm cov qhab nia thiab thauj cov phiaj xwm uas tshwm sim los ntawm PLS tsom xam, raws li qhia los ntawm Mediani li al. [80]. Ib feem tsawg-squares biplot rau cov kev ua ub no radical scavenging (Daim duab 5A) thiab cov khoom tiv thaiv kev laus (Daim duab 5B) tau pom tias tag nrho cov qauv tau sib cais zoo thiab sib cais tsis muaj qhov tseem ceeb outliers. Lub PC1 cais cov nplooj ntawm P. minus, C.caudatus, thiab P. indica los ntawm V. negundo, O.jaoanica, thiab C.longa. Raws li cov kev ua si radical scavenging thiab anti-aging khoom ntawm biplots, tus qauv qhia zoo zog (R'Y) qhov tseem ceeb ntawm {{10}}.988 thiab 0.966, raws li. Lub caij no, qhov kwv yees kwv yees (Q4) rau cov kev ua si radical scavenging thiab cov khoom tiv thaiv kev laus nyob ntawm 0.984 thiab 0.951, feem.

Raws li pom los ntawm PLS biplot ntawm radical scavenging kev ua ub no (Daim duab 5A), cov bioactivities (DPPH, ABTS, thiab ORACassay) tau coj mus rau qhov zoo ntawm biplot, uas yog qhov chaw nquag tshaj plaws thiab ze tshaj rau P.minus, P. indica thiab C.caudatus. Nyob rau hauv sib piv, V. negundo, O.javanica, thiab C. longa tau raug coj mus rau qhov tsis zoo sab ntawm lub biplot, uas tau suav hais tias yog qhov tsawg tshaj plaws cov cheeb tsam thiab tau ntxiv los ntawm DPPH, ABTS, thiab ORAC kev soj ntsuam, thiab pom qhov tsis zoo correlation. nrog bioactivities. Hauv qhov xwm txheej no, P. mimus, P. indica, thiab C.caudatus tau sib cais sib nrug los ntawm cov tshuaj ntsuab tsawg tshaj plaws, qhia tias cov tshuaj ntsuab tau nthuav tawm cov nyhuv radical-scavenging muaj zog, yog li qhia tias cov tshuaj ntsuab no tuaj yeem muaj cov phenolic ntau dua. tshuaj sib xyaw. Ntawm peb cov tshuaj nquag tshaj plaws, P. minus tau pom tias muaj kev sib raug zoo nrog DPPH thiab ABTS kev soj ntsuam, ua raws li ORAC kev soj ntsuam.

Qhov kev tshawb pom no tau pom zoo nrog cov txiaj ntsig hauv vitro ntawm radical scavenging cov haujlwm uas tau ua. Cov txiaj ntsig tau pom tias P. rho tawm tau nthuav tawm lub zog dawb radical scavenging nyhuv piv rau lwm yam tshuaj ntsuab. Cov txiaj ntsig li no tau qhia tias P. minus yog cov tshuaj ntsuab tshaj plaws rau cov tshuaj tiv thaiv cov pa oxygen reactive. Qhov tseem ceeb thib ob metabolites uas tau pab txhawb rau kev ua haujlwm ntawm kev tshem tawm ntawm P. minus tau pom tias yog quercetin, quercetin-3-O-rhamnoside, catechin, isorhamnetin, astragalin, thiab apigenin.oteflavonoidTag nrho cov metabolites no tau nyob ze rau P. rho tawm thiab tseem mus rau DPPHand ABTS radical scavenging assays. Kev kawm yav dhau los los ntawm Mediani li al.[80] Kuj tseem pom tau tias cov qauv khov-qhuav ntawm cov nroj tsuag pom muaj ntau dua ntawm -glucose, -glucose, catechin, thiab chlorogenic acid, uas tuaj yeem ua rau muaj zog DPPH radical scavenging peev ntawm cov tshuaj ntsuab. Txawm li cas los xij, lub siab radical scavenging nyhuv ntawm P.minus kuj yuav pab tau los ntawm cov unidentified metabolites nyob rau hauv lub extract.

Cov kev tshawb pom zoo sib xws kuj tau pom rau cov khoom tiv thaiv kev laus. Daim duab 5B nthuav qhia cov biplot tau los ntawm PLS ntawm cov khoom tiv thaiv kev laus. Cov bioactivities (anti-elastase thiab anti-collagenase kev ua ub no) tau npaj rau ntawm qhov zoo ntawm biplot, uas yog thaj chaw nquag tshaj plaws thiab ze rau P.minus, P. indica, thiab C.caudatus. Hauv qhov sib piv, V.negundo, O.javanica, thiab C.longa tau raug coj mus rau qhov tsis zoo ntawm cov biplot, uas tau suav tias yog qhov chaw ua haujlwm tsawg tshaj plaws thiab tau nyob deb ntawm cov kev ua ub no los tiv thaiv elastase thiab anti-collagenase. Qhov no tau pom qhov tsis zoo lossis tsis muaj zog sib cuam tshuam rau bioactivities. Hauv qhov xwm txheej no, P.mimus, P.indica, thiab C. caudatus tau sib cais los ntawm cov tshuaj ntsuab tsawg tshaj plaws, qhia tias cov tshuaj ntsuab no tau nthuav tawm ntau dua elastase thiab collagenase inhibition. Ntawm peb cov tshuaj nquag tshaj plaws, P. rho tawm dua tau pom tias muaj kev sib raug zoo nrog cov khoom tiv thaiv kev laus thaum piv rau lwm yam tshuaj ntsuab.

Qhov kev tshawb pom no tau pom zoo nrog cov txiaj ntsig hauv vitro ntawm cov khoom tiv thaiv kev laus uas tau ua dhau los. Cov txiaj ntsig tau qhia tias P.minus yog cov tshuaj ntsuab tshaj plaws rau inhibiting elastase thiab collagenase enzymes. Cov metabolites tseem ceeb thib ob uas ua rau muaj kev tiv thaiv kev laus ntawm P. minus tau pom tias yog quercetin, quercetin-3-O-rhamnoside, myricetin derivatives, catechin, isorhamnetin, astragalin, thiab apigenin. Lwm cov metabolites xws li -glucose, -glucose, fumaric acid, thiab fatty acid kuj tseem ua lub luag haujlwm rau bioactivities. Tag nrho cov metabolites no tau nyob ze rau P. minus thiab ua rau cov dej num tiv thaiv elastase thiab anti-collagenase.

Cistanche tuaj yeem tiv thaiv kev laus

Kev ntxub ntxaug ntawm cov tshuaj xaiv no tau pom zoo nrog cov qauv kev ua haujlwm siab radical scavenging tshwj xeeb. minus, P.India thiab C.caudatus, uas tau xav tias yuav tsum tau cais tawm ntawm lwm tus vim lawv cov concentration ntawm cov metabolites theem nrab, tshwj xeeb tshaj yog flavonoids, muaj cov phenolic tebchaw no kuj ntseeg tias yuav pab txhawb kom muaj ntau dua elastase thiab collagenase inhibitory kev ua ntawm cov tshuaj ntsuab no [67-79,89-92].

Kev koom tes ntawm bioactive compounds ntawm cov nroj tsuag rho tawm mus rau dawb radical scavenging peev xwm thiab kev tiv thaiv kev laus tau raug sau tseg los ntawm ntau yam kev tshawb fawb [66,72,75,79,92-101]. Tsis tas li ntawd, cov metabolites xws li flavonoids (quercetin, kaempferol, myricetin, epicatechin, thiab catechin) thiab lwm yam phenols xws li resveratrol thiab procyanidin B2, tau raug pov thawj rau inhibit elastase thiab collagenase [69-71, 79].

Nyob rau hauv txoj kev tshawb no, cov metabolite cov cim qhia txog qhov tseem ceeb ntawm qhov projection (VIP) qhov tseem ceeb tau txheeb xyuas thiab tshuaj xyuas kom tau txais cov metabolites tseem ceeb tshaj plaws uas cuam tshuam nrog kev sim bioactivities. Qhov no tau ua los ua kom muaj kev ncaj ncees ntawm cov txiaj ntsig tau nthuav tawm ntawm no, uas tau tshuaj xyuas qhov muaj peev xwm sib cais ntawm cov txheej txheem metabolites.puritans vitamin cGenerally, the metabolites signal with VIP>{{0}}.5 raug muab coj los txiav txim siab rau qhov tseem ceeb rau kev ntxub ntxaug [102]. Nyob rau hauv txoj kev tshawb no, tag nrho cov metabolites pab txhawb rau cov radical scavenging kev ua ub no thiab cov khoom tiv thaiv kev laus tuaj yeem raug cais raws li cov metabolites tseem ceeb vim lawv cov txiaj ntsig VIP siab dua 1.0 (Table 2). Ob tus qauv PLSbiplots tau lees paub siv 100 qhov kev hloov pauv tsis zoo, kom paub meej qhov siv tau (R2) thiab kev kwv yees (Q2) peev xwm ntawm tus qauv qub nrog ntau tus qauv, piv rau qhov zoo ntawm kev haum. R2 piav qhia tias tus qauv kev nyab xeeb yog qhov tseem ceeb thiab piav qhia qib Y qhov sib txawv hauv cov qauv, thaum Q2 muab cov qauv kev kwv yees zoo ib yam li cov lus qhia los ntawm Eriksson li al. [103].

Generally, when the values of R2 and Q² are nearing 1, it reflects an improved presentation of the model in relation to goodness of fit and predictive quality [80]. In this study, R2 and Q2 values for both of the PLS models fell in the range of 0.951-0.988, which indicated outstanding goodness of fit(R2Y(cum)>0.8)and superior predictive ability (Q2(cum)>0.8). Cov txiaj ntsig tau qhia tias tag nrho cov Y-axis cuam tshuam ntawm R< and="" q-="" for="" the="" assays="" in="" radical="" scavenging="" activities="" and="" anti-aging="" properties="" were="" within="" the="" limits="" of=""><0.3 and=""><0.05.>sibQhov R2 thiab Q2 cuam tshuam qhov tseem ceeb nyob hauv thaj tsam ntawm 0.0367-0.0872 thiab -0.444 txog -0.492, raws li, qhia tias ob qho qauv PLS siv tau thiab tsis qhia overfit. Yog li, ob tus qauv PLS no tuaj yeem raug cais raws li cov qauv kev ua tau zoo thiab cov kev tshawb pom no ua rau muaj kev ntseeg siab ntawm cov qauv.

2.7. Relative Quantification ntawm Secondary Metabolites

Cov txheeb ze ntawm qee cov metabolites thib ob uas tau txheeb xyuas los ntawm cov tshuaj ntsuab xaiv tau pom hauv daim duab 6. Cov metabolites no tau pom ntau dua hauv cov tshuaj nquag tshaj plaws xws li P. minus, tau nyob rau ntawm qhov zoo ntawm biplots, thiab tau nyob ze rau yuav luag tag nrho cov bioactivities kuaj.cistanche yog dab tsiCov txiaj ntsig no tau qhia tias cov metabolites theem nrab tshwj xeeb tshaj yog los ntawm pawg flavonoid ntawm cov tshuaj uas muaj nyob rau hauv cov nyiaj ntau hauv P. minus tej zaum yuav ua rau cov dawb radical eradicator thiab anti-aging zog ntawm cov tshuaj ntsuab no?

Thaum muab piv rau cov qauv sib txawv ntawm cov flavonoid uas tuaj yeem pab txhawb rau lawv txoj kev ua tau zoo, nws tau sau tseg tias cov qauv hydroxylation hauv B-nplhaib yuav yog ib qho tseem ceeb rau kev cuam tshuam ntawm cov metabolites ntawm kev laus enzymes [80] . Sim et al[104] kuj tau qhia tias ntawm ob qib protein thiab mRNA, cov nyhuv inhibitory ntawm cov flavonoids tau los ua muaj zog nrog ntau pawg hauv B-ring, thiab lawv tau tshuaj xyuas cov qauv-kev koom tes ntawm qee cov flavonoids hauv MMP-1 gene qhia hauv UV-Airradiated human dermal fibroblasts.

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Tshuaj thiab Reagents

Deuterated methanol-d4 (CH3OH-d4), non-deuterated KH2PO4, sodium deuterium oxide (NaOD), trimethyl silyl propionic acid-d4 sodium ntsev (TSP), ethanol, thiab methanol tau muab los ntawm Merck Millipore International (Darmstadt, Lub teb chaws Yelemees). Quercetin, phosphate buffer, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) [ABTS ],Trolox,potassium persulfate,2,2'-azobis(2-amidinopropane)[AAPH, epigallocatechin gallate (EGCG), HEPES tsis, elastase enzymes, N-methoxy succinyl-Ala-Ala-Pro-Chloro, N -Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide thiab deuterium oxide (D2O) tau muab los ntawm Sigma (Aldrich, Lub teb chaws Yelemees).

3.2. Cog Khoom thiab Sampling

Cov nplooj tshiab ntawm O.jacanica, P. rho tawm, thiab C.longa tau sau los ntawm Felda Sungai Koyan Satu, Raub, thiab Pahang. V.negundo nplooj tau txais los ntawm lub koom haum ntawm Bioscience, Universiti Putra Malaysia. Cov nplooj tshiab ntawm P. indica tau muab sau rau ntawm University Agriculture Park, Universiti Putra Malaysia, thiab cov nplooj tshiab ntawm C. caudatus nplooj raug sau ntawm Agricultural Institute, Serdang, Selangor. Ib daim ntawv pov thawj ntawm cov tshuaj ntsuab no tau muab tso rau hauv herbarium, Lub Tsev Haujlwm ntawm Bioscience, Universiti Putra Malaysia, thiab txhua tus qauv tau raug lees paub los ntawm tus kws kho tsiaj.Anti aging cistancheTag nrho cov nplooj tau sau tsis tu ncua thaum sawv ntxov, hnub ci hnub kom ntseeg tau tias cov ntsiab lus metabolites. Cov phiaj xwm ntawm qhov chaw qhib rau txhua cov tshuaj ntsuab tau muab cais ua rau rau qhov chaw thiab txhua tus qauv tau sau los ntawm txhua ntu ua rau rau qhov rov ua dua.

3.3.Sample Npaj

Thaum sau, cov nplooj tshiab tau ntxuav hauv qab dej ntws kom tshem tawm tag nrho cov khoom seem, qhuav nrog cov ntaub so ntswg hauv chav kuaj, thiab tam sim ntawd khov nrog kua nitrogen kom nres tag nrho cov tshuaj enzymatic thiab khaws cia cov metabolites ua ntej lyophilization. Cov qauv no tau qhuav rau hauv LABCONCO (Kansas City, MO, USA) lub tshuab ziab khaub ncaws kom qhuav kom txog thaum qhov hnyav thiab noo noo mus txog qis dua 10 feem pua. Tag nrho cov qauv qhuav tau muab tso rau hauv av siv lub tshuab ntxhua khaub ncaws rau cov hmoov zoo thiab sieved siv lub tshuab kuaj sieve (ENDECOTTS LTD.London, England) qhov loj 300 um kom tau txais qhov loj me. Cov hmoov coj mus kuaj tau lub tshuab nqus tsev ntim rau hauv ib lub ntim txhuas los tiv thaiv lawv los ntawm lub teeb raug thiab av noo thiab khaws cia ntawm -80 degree ua ntej kev tshuaj ntsuam.

3.4. Kev rho tawm

Cov txheej txheem rho tawm tau piav qhia los ntawm Mediani li al. [105] tau ua raws nrog qee qhov kev hloov kho. Luv luv, 10 g ntawm txhua cov hmoov coj mus rau hauv 100 mL 60 feem pua ​​​​ethanol nyob rau hauv lub amber conical hwj thiab sonicated rau 1 h siv lub ultrasonic da dej sonicator (WiseClean, qauv WUC-D10H, Seoul, Kauslim) nyob rau hauv kev tswj kub (hauv qab 40 degree) . Cov qauv raug lim los ntawm Whatman lim ntawv no.1 thiab cov seem tau rov muab rho tawm ob zaug thiab lim tom qab thawj qhov kev rho tawm tiav. Tom qab ntawd cov extracts tau muab sib xyaw thiab sib xyaw nrog cov evaporator teb hauv qab lub tshuab nqus tsev ntawm 40 degree. Cov khoom viscous uas tau muab los yog khov-qhuav siv LABCONCO lub tshuab ziab khaub ncaws kom ntseeg tau tias muaj kev tshem tawm cov dej thiab khaws cia ntawm -80 degree kom txog thaum siv ntxiv. Thaum kawg, cov hmoov qhuav qhuav tau diluted mus rau qhov tsim nyog concentrations rau tag nrho cov kev tshawb fawb ua.

3.5. DPPH Radical Scavenging Activity

Cov kev ua si radical scavenging ntawm cov qauv tau txiav txim los ntawm kev ua raws li cov txheej txheem tsim los ntawm Kong li al.[106], uas tau tsim los ntawm kev hloov kho ntawm Brand-Williams li al. [107] nrog kev hloov kho me ntsis. Luv luv, 50uL cov qauv rho tawm hauv methanol ntawm qhov sib txawv (0 txog 500 ug / mL) tau ntxiv nrog 195uLfreshly npaj0.2mMmethanolic2, 2-diphenyl-1-picrylhydrazyl (DPPH) daws thiab khaws cia. Alltestsamples tau npaj nyob rau hauv 96 wellplate. Cov txheej txheem decolourizing tau sau tseg ntawm 515 nm siv lub spectrophotometer (Biotek EL 800 microplate nyeem ntawv, Bio-Tek, Winooski, VT, USA) tom qab 60 min incubation nyob rau hauv qhov tsaus ntuj thiab piv rau kev tswj zoo thiab cov qauv dawb paug. Qhov feem pua ​​​​ntawm radical scavenging kev ua si tau ntsuas raws li qhov sib npaug hauv qab no:

Qhov twg A tswj yog lub absorbance ntawm kev tswj tsis muaj cov nroj tsuag rho tawm thiab Yooj yim yog absorbance ntawm cov nroj tsuag rho tawm.

Cov nroj tsuag rho tawm los yog qhov zoo tswj cov concentrations uas scavenged 50 feem pua ​​ntawm ruaj khov dawb radical DPPH raug xam raws li cov ICs siv cov linear graph ntawm radical scavenging feem pua ​​tiv thaiv.

cog extracts/zoo tswj concentrations. ICso qis qhia tias muaj kev ua haujlwm antioxidant ntau dua. Txhua qhov kev sim tau khiav hauv 6 qhov rov ua dua nrog Trolox thiab quercetin raws li kev tswj hwm zoo.

3.6.ABTS Radical Scavenging Assay

Rau ABTS radical scavenging assay, kev tshuaj xyuas tau ua raws li cov txheej txheem piav qhia los ntawm Arnao li al.[108] nrog qee qhov kev hloov kho. Cov tshuaj npaj tau npaj yog 7 mM ABTS ntxiv rau cov tshuaj thiab 2.45 mM potassium persulfate tov. Txhawm rau npaj cov kev daws teeb meem, ob qho kev daws teeb meem tau muab sib xyaw rau hauv tib qhov ntau thiab sab laug kom hnov ​​​​mob hauv qhov tsaus ntuj rau 16 teev ntawm chav tsev kub. Tom qab ntawd, cov tshuaj ua haujlwm tau diluted nrog dej distilled kom tau txais qhov nqus ntawm 0.700±0.005 units ntawm 734 nm siv lub spectrophotometer (UV-1650PC spectrophotometer, Shimadzu, Kyoto, Nyiv) thiab hu ua ABTS plus solution. Cov tshuaj ABTS ntxiv tau npaj tshiab rau txhua qhov kev xeem. Qhov no ABTS ntxiv tov (900 μL) tau tso cai rau hnov ​​​​mob nrog 100 μL ntawm cov tshuaj ntsuab rho tawm rau 2 min. Tom qab ntawd cov absorbance tau nyeem ntawm 734 nm siv lub spectrophotometer. Tus qauv nkhaus suav nrog 3.1 ug / mL rau thiab 50 ug / mL Trolox tau tsim, thiab cov txiaj ntsig tau qhia tias mg Trolox sib npaug Antioxidant Capacity / g qauv (mg TEAC / g qauv).

3.7. ORAC Radical Scavenging Assay

Kev ntsuam xyuas ORAC los ntsuas qhov ua tau zoo ntawm peroxyl radical scavenging tau ua raws li tau hais los ntawm Huang li al. [109] siv FLUOstar OPTIMA microplate fluorescence nyeem ntawv (BMG LABTECH, Ortenberg, Lub teb chaws Yelemees). Txhua cov tshuaj ntsuab extract thiab Trolox (tus qauv) tau npaj hauv 75 mM phosphate buffer solution (PBS) pH 7.4. Hauv 96-zoo dub microplate, tag nrho ntawm 150 μL fluorescein (10 nM yaj hauv PBS) tau ntxiv tom qab 25 μL ntawm Trolox, cov nroj tsuag rho tawm, lossis PBS ua ib qho dawb paug. Cov kev daws teeb meem no tau muab tso rau hauv qhov dej triplicate. Lub microplate tau incubated rau 15 feeb ntawm 37 degree thiab npog nrog lub hau. Lub fluorescence tau ntsuas nrog excitation wavelength ntawm 458 nm thiab emission wavelength ntawm 520 nm. Lub teeb liab keeb kwm yav dhau tau txiav txim siab los ntawm kev ntsuas txhua 90s.

Tom qab ntawd, 25 uL tshiab tau npaj 2,2'-azobis (2-amidinopropane) (AAPH, 240 mM hauv PBS) tau qhia los ntawm on-board injectors. Qhov kev lwj ntawm fluorescence tau coj mus txog 90 min siv tib lub zog thiab emission wavelengths. Kev ntsuam xyuas tau ua tiav rau thaj chaw hauv qab nkhaus rau cov qauv (fluorescence piv rau lub sijhawm) rho tawm thaj tsam hauv qab qhov nkhaus rau qhov khoob thiab muab piv rau tus qauv nkhaus (25-400 μM Trolox). Cov txiaj ntsig ORAC ntsig txog Trolox tau suav nrog kev sib npaug raws li hauv qab no:

3.8.Elastase Inhibition Assay

Cov kev ua elastase inhibitory tau txiav txim siab raws li cov txheej txheem piav qhia los ntawm Krause li al.[110], nrog kev hloov kho me me los ntawm Ndlovu li al. [75]. Cov qauv qhov dej muaj 25 μL 0.1 M HEPES buffer (pH7.5), 25 μL tshuaj ntsuab extract (100 ug / mL), thiab 25 μL elastase enzyme (1 ug / mL). Lub qhov dej dawb xwb tsuas muaj 75 μL HEPES tsis thiab cov qhov dej tswj tsis zoo muaj 50 μL HEPES tsis thiab 25 μL elastase enzyme. Qhov zoo tswj qhov dej muaj 25 μL HEPES tsis, 25 μL N-methoxy succinyl-Ala-Ala-Pro-Chloro (10 ug / mL), thiab 25μL ntawm elastase enzyme. Cov kuab tshuaj tswj qhov dej muaj 25 μL HEPES tsis, 25 μL ntawm 10 feem pua ​​​​methanol, thiab 25 μL elastase enzyme. Kev tswj tsis pub dawb rau cov tshuaj ntsuab extract (rau xim tswj ntawm txhua qhov kev sim extract) muaj 150 μL HEPES tsis muaj thiab 25 uL ntawm cov tshuaj ntsuab extract. Tom qab ntawd lub micro-zoo phaj yog incubated rau 20 min ntawm chav tsev kub. Tom qab no, 100μL substrate (N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, 1 mM) tau ntxiv. Tom qab ntawd cov phaj tau incubated rau 40 feeb ntxiv ntawm 25 degree. Tom qab incubation, lub absorbance tau nyeem siv lub spectrophotometer (Biotek EL800microplate nyeem) ntawm 405 nm. Qhov feem pua ​​inhibition ntawm cov tshuaj ntsuab rho tawm tau suav nrog kev sib npaug raws li hauv qab no:

Qhov twg A tswj yog qhov absorbance ntawm tsis nrog elastase thiab hnyav thiab Atest yog absorbance ntawm tsis, elastase, thiab tshuaj ntsuab extract los yog N-Methoxysuccinyl-Ala-Ala-Pro-Chloro.

3.9. Collagenase Inhibition Assay

Kev ua haujlwm ntawm collagenase inhibition tau ua raws li txoj kev ntawm Van-Wart thiab Steinbrink [111] (1981) nrog kev hloov kho los ntawm Madrone li al. [92]. Qhov kev sim tau ua tiav hauv 50 mM TES tsis (pH7.4 nrog 0.36 mM CaCl2). Collagenase enzyme los ntawm Clostridium histolyticum (ChC-EC.3.4.23.3) tau npaj ntawm qhov concentration ntawm 0.8 units/mL (solved nyob rau hauv TES buffer stock solution). Cov hluavtaws substrate N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) tau npaj ntawm qhov concentration ntawm 2 mM nyob rau hauv TES buffer stock solution. Tag nrho cov ntim ntawm 150 μL rau cov tshuaj tiv thaiv zaum kawg muaj 46.3 μL TESbuffer, 60 μL FALGPA (0.8 mM FALGPA kawg concentration), 18.7 μL collagenase enzyme (0.1 units / mL kawg concentration), thiab 25μL tshuaj ntsuab rho tawm 100 ug/ml). Cov tshuaj ntsuab rho tawm thiab cov enzymes hauv TES tsis tau raug muab tso rau hauv chav sov li 15 feeb ua ntej ntxiv cov substrate los pib cov tshuaj tiv thaiv. Kev tswj tsis zoo tau ua tiav nrog TES tsis muaj. Tom qab ntxiv cov substrate, lub absorbance tau nyeem tam sim ntawd ntawm 340 nm siv lub spectrophotometer (Benchmark Plus Microplate, Bio-Rad 170-6930, Bio-Rad, Hercules, CA, USA) nyob rau hauv 96 zoo microtiter daim hlau thiab tsis tu ncua ntsuas rau lwm tus. 20 feeb. Kev tswj tau zoo tau ua tiav siv epigallocatechin gallate (EGCG) ntawm 12.5ug / mL. Qhov feem pua ​​inhibition ntawm cov qauv tau suav nrog kev sib npaug raws li hauv qab no:

qhov twg A tswj yog qhov absorbance ntawm TES tsis thiab Atest yog absorbance ntawm TES tsis, collagenase enzyme, thiab cog extract los yog FALGPA.

3.10.Metabolite Profiling Siv 'H-NMR Kev Ntsuas

Cov metabolite profiling siv H-NMR ntawm cov tshuaj xaiv tau ua raws li cov txheej txheem piav qhia los ntawm Kim li al. [47] nrog kev hloov kho me ntsis. Crude tshuaj ntsuab rho tawm (25mg) tau pauv mus rau hauv mL microcentrifuge raj. Kev sib xyaw ntawm methanol-d4 thiab KHPO4 tsis nyob hauv Dao (pH 6.0) uas muaj 0.1 feem pua ​​trimethyl silypropionic acid sodium ntsev (TSP) tau ntxiv rau cov qauv tshuaj ntsuab nrog tag nrho cov ntim ntawm { {10}}.7mL ntawm (1:1) ratio. Cov hlab microcentrifuge uas muaj cov tshuaj ntsuab rho tawm ces vortexed rau 1 min ntawm chav tsev kub ua raws li ultrasonication rau 15 min. Tom qab ntawd, qhov sib tov tau centrifuged rau 10 min ntawm 5678g kom cais cov supernatant los ntawm cov ntaub ntawv tsis tuaj yeem. Tom ntej no, 0.6mL ntshiab supernatant tau muab tso rau hauv NMR hlab thiab raug rau 'H-NMR tsom xam. Kev tsom xam ntawm 'H-NMR tau ua tiav ntawm 500 MHz Varian INOVA NMR spectrometer (Varian Inc., Palo Alto, CA, USA) uas ua haujlwm ntawm qhov zaus ntawm 499.887 MHz thiab spectra tau kaw ntawm 26 degree. Txhua qhov spectrum suav nrog 64 scans nrog 3.53 min ntawm lub sijhawm tau txais thiab qhov dav ntawm 20 ppm. Cov ntaub ntawv tau txheeb xyuas siv Chenomx software(v.6.2)(Clhenomx Inc, Edmonton, AB, Canada) los ua phasing thiab hauv paus kev kho nrog kev teeb tsa zoo ib yam. Kev txheeb xyuas cov ntaub ntawv sib txawv tau ua los ntawm SIMCA software (version 13.0, Umetrics, Umea, Sweden). 3.11. Bucketing ntawm 'H-NMR Spectra

Bucketingof H-NMR spectra tau ua los ntawm kev siv Chenomxsoftware(v.6.2, Edmonton, AB, Canada). Tag nrho cov spectra tau muab tso rau hauv 0 5-10 0 pprn cheeb tsam nrog tib qhov tsis muaj. Cov tsis suav nrog qhov dav spectral ntawm §0.04 uas tau txais tag nrho ntawm 245 thaj chaw sib koom ua ke rau txhua qhov NMR spectrum. Cov tshuaj hloov pauv rau cov dej thiab cov seem methanol-d ntawm δ4.70-4.90 thiab δ3.27-3.35 feem raug tshem tawm. Cov ntaub ntawv sib xyaw ua ke yog tom qab ntawd raug rau cov ntaub ntawv sib txawv (MVDA).

3.12. Relative Quantification ntawm metabolites

Cov metabolites uas tau txheeb xyuas tau raug tshuaj xyuas rau lawv cov txheeb ze sib txheeb uas tau xam raws li qhov nruab nrab ntawm thaj tsam ntawm cov teeb liab tom qab binning ntawm LH-NMR spectra.

3.13. Kev txheeb cais

Tag nrho cov txiaj ntsig ntawm rau qhov kev hloov pauv tau qhia raws li qhov txhais tau tias ± tus qauv sib txawv. Rau kev ntsuas ntawm radical scavenging kev ua ub no, inhibition ntawm elastase thiab collagenase kev ua ub no, thiab txheeb ze ntawm cov metabolites, statistical analyses tau ua siv Minitab 16 (Version 16, Minitab Inc., State College, PA, USA). Kev soj ntsuam ntawm qhov sib txawv (ANOVA) tau siv los tshuaj xyuas qhov sib txawv tseem ceeb ntawm txoj kev nrog p<0.05 considered="" as="" significantly="" different.="" principal="" component="" analysis="" (pca)="" and="" partial="" least="" square="" (pls)="" from="" the="" multivariate="" data="" analysis="" （mvda）,="" were="" implemented="" using="" simca-p="" software（v.="" 13.0,="" umetrics,="" umeå,="" sweden）using="" the="" pareto="" scaling="" method="" after="" the="" binning="" of="" nmr="" spectra="" was="" completed.="" the="" correlation="" between="" metabolites="" components="" and="" ics0="" values="" for="" dpph="" radical="" scavenging="" activity="" was="" converted="" to="" 1/ic5so="" in="" order="" to="" acquire="" the="" same="" trend="" as="" the="" functional="" properties="">

4. Cov lus xaus

Daim ntawv thov ntawm H-NMR kev soj ntsuam ua ke nrog MVDA tau ua tiav hauv kev tshawb nrhiav qhov hloov pauv hauv cov metabolites ntawm cov tshuaj ntsuab xaiv. Cov qhab-nees PCA tau pom qhov sib cais ntawm cov tshuaj ntsuab raws li lawv pawg. Txoj kev tshawb no tau nthuav tawm tias P. minus muaj cov txiaj ntsig zoo tshaj plaws ntawm kev tshem tawm los ntawm DPPH thiab ABTS kev soj ntsuam thiab nthuav tawm cov dej num tiv thaiv kev laus siab tshaj plaws. Ob qhov kev ntsuam xyuas ntawm PLS tau ua pov thawj ntxiv qhov txiaj ntsig no, vim tias muaj kev sib koom ua ke muaj zog tau pom ntawm cov metabolites pom hauv P. rho tawm thiab kuaj bioactivities. Cov metabolites nquag ntseeg tias yuav pab txhawb rau cov kev ua si radical scavenging thiab tiv thaiv kev laus ntawm P.minus muaj xws li quercetin, quercetin-3-O-rhamnoside, myricetin derivatives, catechin, isorhamnetin, astragalin, thiab apigenin. Yog li, nws tuaj yeem xav tias cov metabolites no yog lub luag haujlwm rau lub zog radical scavenging nyhuv thiab siab tiv thaiv kev laus ntawm P. minus. Cov metabolites no feem ntau yog los ntawm pawg flavonoids, tshwj xeeb tshaj yog flavonols. Yog li ntawd, nws tuaj yeem pom tau tias cov metabolites los ntawm P. minus tuaj yeem yog ib qho kev cog lus dawb radical eradicator thiab aging enzymes inhibitor uas tuaj yeem siv rau ncua kev laus cov tsos mob thiab kev kho mob ntawm cov kab mob aging-associated chronic disease. Cov kev tshawb pom no yuav pab tsim kom muaj lub zog ntawm P. rho tawm raws li qhov muaj peev xwm ntawm cov tshuaj tiv thaiv kev laus thiab ua cov dawb radical eradicator.

Kab lus no tau muab rho tawm los ntawm Molecules 2019, 24, 3208; doi: 10.3390/molecules24173208 www.mdpi.com/journal/molecules