Pingping Zou

Pingping Zoua, †, Yuelin Songb, †, Wei Leia, Jun Lib, Pengfei Tua, b,

Yong Jiang, ⁎

Acetate Key Laboratory of Natural and Biomimetic Drugs, Tsev Kawm Ntawv Pharmaceutical Sciences, Peking University, Beijing 100191, Tuam Tshoj niaj hnub tshawb fawb Center for Traditional suav tshuaj, Tsev Kawm Ntawv ntawm Suav Materia Medica, Beijing University of suav tshuaj, Beijing 100029, Tuam Tshoj

Tau txais 17 Tsib Hlis 2017; hloov kho 20 Lub Rau Hli 2017; tau txais 17 Lub Xya Hli 2017

Abstract: Cistanche deserticola(CD) yog ib qho ntawm ob qhov kev tso cai los ntawm cov nroj tsuagCistanches Herba, ib tug zoo-paub tshuaj nroj tsuag. Ntawm no, 1H NMR spectroscopy tau ua haujlwm los ua tus yam ntxwv ntawm cov tshuaj lom neeg thiab paub qhov txawv ntawm qhov sib txawv, nrog rau kev tawm tswv yim ua haujlwm tshiab rau CD. Lub teeb liab txoj haujlwm tau ua tiav los ntawm ntau qhov ib thiab ob-seem NMR spectroscopic cov txheej txheem ua ke nrog cov ntaub ntawv muaj thiab cov ntsiab lus tseeb. Qhov chaw sab saud ntawm cov nroj tsuag tau txawv ntawm qhov qis los ntawm kev sib txuas ntawm 1H NMR spectroscopic dataset nrog ntau qhov kev txheeb xyuas txheeb cais. Ib txoj kev ua haujlwm tshiab uas hyphenated steaming nrog khov-qhuav tau pom tias yuav zoo dua rau kev sib xyaw ua ke nrog qhov cub-qhuav lossis ncaj qha khov-drying ntawm holistic 1H NMR-based metabolomic characterization. Phenylethanoid glycosides, feem ntau echinacoside thiabacteoside, tau raug tshuaj xyuas thiab lees paub tias yog cov cim tshuaj ua lub luag haujlwm rau kev nthuav tawm qhov zoo tshaj plaws ntawm kev ua haujlwm tshiab, whereas serial thawj metabolites, tshwj xeeb tshaj yog cov carbohydrates thiab tricarboxylic acid cycle metabolites, tau pom tias yog thawj cov molecules tswj kev ntxub ntxaug ntawm sab sauv thiab sab qis. tsob nroj. Sib sau ua ke, 1H NMR spectroscopy tau pom tias yog cov cuab yeej tshuaj ntsuam ntau yam los ua tus yam ntxwv ntawm cov tshuaj lom neeg thiab coj mus rau qhov tob ntawm kev siv CD los ntawm kev muab cov ntaub ntawv zoo thiab ntau yam.

KEEB KWM:Cistanchedeserticola; 1H NMR-raws li metabolomics; Kev ua haujlwm ua haujlwm; Qhov sib txawv;Phenylethanoid glycoside; Tricarboxylic acid cycle metabolites;Echinacoside; Acteoside

Yog xav paub ntxiv thov hu rau:Joanna.jia@wecistanche.com

Cistanche deserticola muaj ntau yam teebmeem, nyem qhov no kom paub ntau ntxiv

1. Taw qhia

Cistanches Herba(CH, Suav lub npe: Roucongrong), pib archived hauvShen Nong's Suav Materia Medica, tau raug suav hais tias yog ib qho ntawm cov khoom noj muaj txiaj ntsig zoo tshaj plaws thiab cov nroj tsuag tshuaj, thiab tau txais txiaj ntsig zoo li "Ginseng ntawm cov suab puam"^1,2. Raws li ib qho ntawm ob qhov chaw cog qoob loo ntawm CH,Cistanche deserticola(CD, Orobanchaceae) yog cov nroj tsuag holoparasitic thiab feem ntau faib rau sab qaum teb thiab sab hnub poob ntawm Tuam Tshoj.^2,3. Nws tau siv dav rau kev kho mob ntawm lub raum tsis zoo uas ua rau lub cev tsis muaj zog, mob hauv lub duav thiab lub hauv caug, poj niam tsis muaj menyuam, thiab cem quav hauv cov tshuaj suav tshuaj rau ntau pua xyoo.^3–5. Txawm li cas los xij, cov tsiaj qus ntawm CD yog nyob ntawm ntug ntawm kev ploj tuag nyob rau xyoo tas los no vim kev sau qoob loo ntau dhau, thiab nws tau raug teev tseg tias yog ib qho ntawm cov nroj tsuag hauv chav II xav tau kev tiv thaiv hauv Suav teb.¹. Ntxiv mus, CD muab ib qho tseem ceeb pab rau kev tswj suab puam. Yog li ntawd, nws yog ib qho tseem ceeb tab sis nyuaj rau siv cov khoom siv tshuaj ntsuab no kom zoo dua.

Kev tshawb fawb txogCistanchecov nroj tsuag pib thaum xyoo 1980s⁶. Kev tshawb nrhiav phytochemical qhia pom tias muaj ntau hom tshuaj lom neeg,e.g., phenylethanoid glycosides (PhGs), iridoids, lignans, fatty acids, alditols, thiab carbohydrates, hauv CD⁷. Ntawm lawv, PhGs feem ntau hais txog vim lawv qhov dav dav ntawm kev ua haujlwm lom neeg, suav nrog kev tiv thaiv oxidation, tiv thaiv kev laus, tiv thaiv qaug zog, tiv thaiv kab mob, txhim kho lub cev tiv thaiv kab mob, txhim kho kev kawm thiab nco txog Alzheimer's kab mob nas, thiab lwm yam.¹. Tsis ntev los no, PhGs tau ntxim nyiam ntau dua li cov neeg tuaj yeem siv tshuaj tshiab los kho cov kab mob neurodegenerative. Tshwj xeeb, kev sib sau ua ke ntawm tag nrho cov PhGs pom hauv CH tau tsim los ua cov tshuaj tshiab, sau npe tag nrho.Cistancheglycosidecapsule (Memoregain^®) rau kev kho mob vascular dementia⁸. Echinacoside, cov khoom muaj txiaj ntsig zoo tshaj plaws ntawm tag nrho cov PhGs nthuav tawm cov teebmeem los tiv thaiv apoptotic ntawm SHSY5Y neuronal hlwb tom qab TNF-induced apoptosis thiab thim rov qab tsis txaus hauv Parkinson tus kab mob nas.⁹. Ntxiv mus, lwm qhov tseem ceeb tshaj plaws, acteoside (tseem hu ua verbascoside) tuaj yeem tiv thaiv apoptosis hauv neurons.¹⁰los tiv thaiv neurotoxicity hauv PC12 hlwb induced los ntawm 1-methyl-4-phenylpyridium los yog glutamate¹¹, thiab txhim kho scopolamine-induced nco tsis txaus².

Zoo ib yam liCordycepsthiabGinseng, CD tau siv dav thiab muaj nuj nqis ua zaub mov noj qab haus huv. Cov txiaj ntsig tiag tiag thiab pom tau ntawm CD zoo li yuav ua lub luag haujlwm txiav txim siab rau tus nqi ntawm cov tshuaj crude. Muab lub cev loj ntawm cov tshuaj phem ntawm CD, cov hlais tau nrov dua hauv kev ua lag luam. Feem ntau, enzymatic inactivation thiab dej deprivation yog ob kauj ruam tseem ceeb thaum lub sij hawm ua cov txheej txheem tshuaj. Cov txheej txheem ziab kom qhuav ntawm CD muaj xws li rwb thaiv tsev, qhov cub-qhuav, salting, thiab cellar cia. Txawm li cas los xij, cov khoom lag luam los ntawm cov txheej txheem no feem ntau raug kev txom nyem los ntawm cov tsos mob tsis zoo thiab cov ntsiab lus tsawg ntawm PhGs, yog li cuam tshuam rau daim ntawv thov dav dav thiab kev siv CD. Xyoo 2007, peb pab pawg tau tshaj tawm cov txheej txheem tshiab rau CD uas tau khaws cia cov ntsiab lus ntawm echinacoside thiabacteosidehauv slices¹⁴. Txawm li cas los xij, txij li cov khoom lag luam los ntawm cov txheej txheem no tseem raug kev txom nyem los ntawm qhov tsis pom kev zoo, peb tam sim no piav qhia txog kev ua cov txheej txheem los txhim kho cov tsos thiab khaws cia cov ntsiab lus ntawm PhGs hauv cov ntaub ntawv ua tiav.

Cov cuab yeej tshuaj ntsuam xyuas tau tam sim no tau siv rau kev tshuaj xyuas tshuaj ntawm CD, suav nrog nyias txheej chromatography, ua haujlwm siab ua kua chromatography (HPLC) ua ke nrog ntau yam khoom siv, xws li diode array detector (DAD), evaporative light scattering detector (ELSD), electron capture detector (ECD), thiab tandem mass spectrometer (MS/MS). PhGs, tshwj xeeb tshaj yog echinacoside thiab acteoside, feem ntau tau txais los ua cov cim zoo¹. Tus ntiv tes ntawm cov tshuaj ntsuab no kuj tau tsim los siv HPLC-DAD thiab HPLC-DAD-MS/MS^15,16. Txawm li cas los xij, nws tseem yog ib qho kev sib tw los ntsuas qhov zoo ntawm CD vim nws cov tshuaj lom neeg tsis tshua muaj zog. Feem ntau hais lus, txoj hauv kev tsom mus rau ntau qhov kev txheeb xyuas tsis muaj peev xwm muab cov tshuaj holistic saib rau cov khoom siv raw, txawm hais tias muaj ntau cov ntaub ntawv zoo thiab muaj nuj nqis tuaj yeem tau txais los ntawm LC-MS / MS. Tsis tas li ntawd, HPLC- cov tswv yim tsom xam muaj feem xyuam tuaj yeem raug txwv los ntawm cov kuab tshuaj ntau, kev npaj ua qauv txaus ntshai, thiab / lossis cov txheej txheem siv sijhawm. Yog li ntawd, ib tug tshiab haum-rau-lub hom phiaj analytical cuab tam muaj peev xwm ntawm yielding tag nrho cov ntaub ntawv ntawm lub compound pas dej ua ke yog yuav tsum tau rau kom pom zoo tshuaj ntsuam xyuas. Hmoov zoo,¹H NMR spectroscopy tau ua kom pom tseeb raws li qhov txaus nyiam "tag nrho hauv ib qho" cov cuab yeej muaj peev xwm muab tsis tau tsuas yog cov ntaub ntawv zoo nkaus xwb tab sis kuj muaj cov ntaub ntawv ntau ntxiv rau ntau yam ntawm ob qho tib si thawj thiab theem nrab metabolites nrog cov qauv yooj yim npaj thiab nrhiav tau sai.^17–20. Txog tam sim no, dav siv ntawm¹H NMR spectroscopy tau raug tsim los rau kev txiav txim siab ib txhij, tshuaj lom neeg, thiab metabolomics ntawm complex matrices.

Txawm hais tias ntau qhov kev tshawb nrhiav tau ua tiav rau cov tshuaj ntsuab muaj txiaj ntsig no, nws cov tshuaj lom neeg thoob ntiaj teb tsis paub ntau. Vim tias CD yog cov kab mob cab, qhov qis qis yuav tsum yog lub luag haujlwm rau kev xa cov khoom noj khoom haus, feem ntau yog cov metabolites, los ntawm tus tswv tsev mus rau sab saud, thaum lub zog muaj zog metabolism, xws li paj, feem ntau tshwm sim nyob rau sab sauv. Yog li ntawd, nws tsim nyog xav tias qhov sib txawv tshwm sim rau cov metabolome ntawm qhov sib txawv. Yog li ntawd, nyob rau hauv txoj kev tshawb no tam sim no, peb lub hom phiaj 1) kom paub meej cov yam ntxwv ntawm cov tshuaj lom neeg ntawm CD siv.¹H NMR spectroscopy ua ke nrog ntau qhov sib txawv (2D) NMR ntsuas; 2) kom paub meej qhov sib txawv ntawm sab sauv thiab sab qis, thiab 3) kom nthuav tawm kev ua haujlwm tshiab los ntawm¹H NMR-raws li kev tshawb fawb metabolic. Cov txiaj ntsig tau txais yuav tsum muab cov lus qhia ruaj khov rau kev siv tshuaj ntsuab ntxiv rau txoj hauv kev zoo dua, tshwj xeeb tshaj yog rau kev ua haujlwm ntawm ntau qhov chaw thiab cov txheej txheem ua haujlwm.

2. Kev sim

2.1. Cov ntaub ntawv cog

Kaum ob pawg ntawm cov ntaub ntawv tshiab (CD1–CD12, Table S1, cov ntaub ntawv ntxiv) tau sau los ntawm Inner Mongolia, Xinjiang, thiab Ningxia autonomous cheeb tsam hauv Suav teb. Cov keeb kwm botanical ntawm tag nrho cov ntaub ntawv nyoos tau raug txheeb xyuas raws li

1H NMR-raws li metabolomics ntawmCistanche deserticola 649C. deserticolalos ntawm ib tus kws sau ntawv, Prof. Pengfei Tu. Txhua daim ntawv pov thawj tau muab tso rau ntawm lub herbarium ntawm Niaj hnub nimno tshawb fawb Center rau tsoos suav tshuaj, Peking University (Beijing, Tuam Tshoj).

2.2. Tshuaj thiab reagents

Methanol-d4 (CD3OD, deuterium abundance, 99.8 atom feem pua ​​D), deuterated dej (D2O, deuterium abundance, 99.8 atom feem pua ​​D), thiab sodium 3-trimethylsilyl [2,2,3,3-d4 ] propionate (TSP-d4) tau txais los ntawm Cambridge Isotope Laboratories (Andover, MA, USA). Kev ntsuas qib methanol tau yuav los ntawm Beijing Chemical Works (Beijing, Suav).

Cov ntsiab lus tseeb, suav nrog echinacoside, acteoside, thiab mannitol, tau lim los ntawm CD hauv peb lub chaw kuaj ua ntej 14, thiab sucrose, -galactose, nrog rau -glucose tau muab los ntawm Sigma–Aldrich (St. Louis, MO, USA). Qhov purities ntawm tag nrho cov ntaub ntawv tau txiav txim siab ntau dua 98 feem pua ​​​​ntawm HPLC-UV thiab 1H NMR kev tshuaj ntsuam.

2.3. Kev npaj ua qauv

Tag nrho cov ntaub ntawv nyoos (CD1-CD11) tshwj tsis yog CD12 raug txiav rau hauv qhov chaw sab saud (GU) thiab qis qis (GD) ntawm nruab nrab ntawm tag nrho cov stems, thiab tag nrho cov seem raug txiav rau hauv nyias nyias (kwv yees li 6- hli. - tuab txhua). Tom qab ntawd, txhua daim ntawm CD1–CD12 tau ua tiav steamed rau 10 min thiab qhov cub-qhuav ntawm 6{{40}} degree rau 48 h kom tau txais cov qauv qauv. Ob txoj kev ua lwm yam, suav nrog kev ua kom qhuav ncaj qha thiab 10-min steaming ua raws li kev ua kom khov-qhuav, tau ua rau qee cov qauv xaiv, suav nrog CD1-GU, CD4-GU, CD4-GD, CD11-GD, thiab CD12, kom them tau ob txheej ntxiv ntawm cov qauv ua tiav (b- thiab c-hom qauv). Cov lus piav qhia ntxaws ntxaws ntawm tag nrho cov qauv yog qhia hauv Table S1. Ua ntej kev rho tawm, tag nrho cov txheej txheem tiav tau raug crushed rau hauv cov hmoov nrog cov qauv zeb (qauv YF102, Ruian Yongli Pharmacy Machinery, Tuam Tshoj) thiab sifted los ntawm 80 mesh sieve. Cov ntaub ntawv cog ntoo pulverized tau raug ntsuas kom raug (kwv yees li 200 mg rau txhua tus) thiab muab rho tawm nrog 50- quav ntawm 50 feem pua ​​aqueous methanol (w/v) hauv da dej ultrasonic (25 degree) rau 40 min. Tom qab ntawd, 50 feem pua ​​aqueous methanol tau ntxiv los them rau qhov poob phaus thaum lub sijhawm rho tawm. Txhua cov extract tau centrifuged ntawm 10, 000 × g ntawm 10 degree rau 10 min thiab lim los ntawm 0.22 μm membrane. Aliquots (2 mL) ntawm supernatant tau tom qab evaporated thiab ntxiv exsiccated siv lub tshuab nqus tsev ziab ntawm 30 degree rau 30 min. Cov seem qhuav ntawm txhua tus qauv tau rov ua dua tshiab siv 0.5 mL sib tov (1: 1, v / v) ntawm CD3OD thiab phosphate tsis nyob hauv D2O uas muaj 0.05 feem pua ​​​​TSP-d4 (pH 7.4), thiab cov tshuaj tau hloov mus rau hauv 5 hli. (id) NMR tube (Norell ST500-7) rau NMR kev soj ntsuam. Txhua tus qauv tau txheeb xyuas hauv triplicate.

2.4. NMR ntsuas

Tag nrho 1H NMR, 13C NMR, thiab 2D NMR spectra tau kaw rau ntawm Varian Unity Plus 500 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) ntawm 499.91 MHz proton zaus nruab nrog TCI cryoprobe thiab Z-gradient system. Qhov tsis zoo ib yam tau siv rau ob qho tib si rho tawm thiab siv cov khoom sib txuas kom tau txais qhov sib piv spectra. Rau 1H NMR kev ntsuas, 256 scans tau txais nrog cov nram qab no tsis: spectra width, 8012.6 Hz (16 ppm); mem tes dav, 11.05 μs (lub kaum sab xis 90 degree); lub sijhawm tau txais, 2.04 s; so ncua (d1), 2 s; thiab qhov kub thiab txias, 298 K. CD3OD tau ua haujlwm txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau nws, cov txheej txheem tshuaj xyuas, cov tshuaj lom neeg cov tshuaj, nws raug nquahu kom siv cov tshuaj tua kab mob los ntawm TSP-d4 ntawm δ 0. Cov txheej txheem ntub dej tau txais kev pom zoo los txwv qhov kev siv ntawm H2O teeb liab uas nyob ib puag ncig δ 3.3021. Ib qho exponential muaj nuj nqi nrog kab broadening (LB) yam raws li 0.3 Hz tau thov, thiab cov ntaub ntawv yog xoom-filled kom tsawg kawg tsib cov ntaub ntawv ntsiab lus saum toj ib nrab-dav rau txhua resonance los lav qhov tseeb thiab txhim khu kev qha kev koom ua ke. Cov teeb liab dawb induction decay (FID) yog Fourier hloov pauv (FT), thiab tag nrho cov spectra tau manually phased thiab kho siv qhov kev pab cuam automated polynomial baseline.

Txhawm rau pab cov tshuaj lom neeg tus yam ntxwv thiab kev teeb tsa,

13C NMR thiab ntau yam 2D ​​NMR analyses, xws li 1H–1H

correlation spectroscopy (1H–1H COSY), heteronuclear single quantum coherence spectroscopy (HSQC), thiab heteronuclear multiple bond correlation spectroscopy (HMBC) kuj tau txais rau tus qauv tus neeg sawv cev (CD1-GUI) siv cov kev pabcuam defaulted. Qhov dav spectral rau COZY yog δ 0.5–10.0 nyob rau hauv ob qhov ntev thiab 128t1 increments rau txhua t1. Kaum rau lub sijhawm hloov pauv siv 1.

Qhov zoo tshaj plaws ib-bond thiab n-bond heteronuclear coupling tas li rau HSQC thiab HMBC yog 146 thiab 8 Hz, feem. Qhov ntau thiab tsawg tau teeb tsa raws li -0.5–10.0 ppm hauv F2 qhov loj me thiab 0–200 ppm hauv F1 qhov loj me, feem.

2.5. Multivariate statistical tsom xam ntawm NMR spectroscopic dataset

1H NMR spectra tau ua tiav siv MestReNova software (version 5.2.5, Mestrelab Research, Santiago de Compostella, Spain). Txhua qhov spectrum raug ntsuas rau tag nrho cov kev siv zog thiab txo mus rau thaj tsam sib npaug ntawm qhov dav sib npaug (0.02 ppm) ntawm thaj tsam ntawm δ 0.50–9.50 tom qab tsis suav cov cheeb tsam ntawm δ 4.70–

5. Kev tsom xam cov ntsiab lus tseem ceeb (PCA) nrog Pareto (Par) scaling as well as orthogonal partial least squares- discriminant analysis (OPLS-DA) with unit variance (UV) scaling were performed with SIMCA-P 12.0 software (Umetrics, Umeå, Sweden).

2.6. Kev txiav txim siab ib txhij ntawm echinacoside thiab acteoside siv HPLC-UV

Txhawm rau txhawm rau txheeb xyuas qhov tseeb ntawm peb txoj kev ua tiav, Agilent 1260 series HPLC system muaj xws li degasser, lub twj tso kua mis quaternary, autosampler, kem qhov cub, thiab diode array detector (Agilent Technologies, Santa Clara , CA, USA) tau ua haujlwm rau kev txiav txim siab ib txhij ntawm echinacoside thiab acteoside hauv txhua qhov qauv a-, b- thiab c-hom qauv. Ib qho Agilent Zorbax SB- C18 kem (250 mm × 4.6 mm, particle loj 5 μm, Agilent Technologies) tiv thaiv los ntawm tus neeg saib xyuas kem (10 mm × 4.6 mm, particle loj 5 μm) tau xaiv los ua chromatographic sib cais. Cov qauv npaj raws tu qauv, theem mobile, thiab elution program ua raws li cov lus piav qhia hauv Suav Pharmacopeia (2010 Edition) 3 nrog kev hloov kho me me. Hauv luv luv, ultrasonic-pab extraction tau ua rau 30 min siv 50 feem pua ​​aqueous methanol; 30 feem pua ​​​​aqueous methanol tau txais yuav los ua lub sijhawm txawb rau isocratic elution ntawm tus nqi ntws ntawm 1.0 mL / min, thiab lub qhov cub tau khaws cia ntawm

30 degree. Kev kuaj pom wavelength tau teem rau ntawm 330 nm, thiab qhov ntim ntim tau teem li 10 μL.

3. Cov txiaj ntsig thiab kev sib tham

3.1. Optimization ntawm extraction thiab spectroscopic tsis

Txhawm rau kom tau txais txiaj ntsig zoo 1H NMR spectra rau tag nrho cov qauv, ntau yam tsis tau ua tib zoo ua tib zoo siv tus qauv sawv cev (CD1-GUI). Ua ntej, cov kuab tshuaj extraction raug xaiv ntawm 30 feem pua ​​aqueous methanol, 50 feem pua ​​aqueous methanol, thiab methanol. Cov txiaj ntsig tau qhia tias cov lus teb ntau dua tau them los ntawm 50 feem pua ​​​​aqueous methanol tshaj li ob tus, pom zoo nrog cov txheej txheem rho tawm pov thawj hauv Suav Pharmacopeia (2015 tsab) 3. Ultrasonic-pab extraction raug xaiv vim nws yooj yim ua hauj lwm ntawm yields piv rau cov kub-reflux extraction thiab Soxhlet extraction (cov ntaub ntawv tsis qhia). Tom qab ntawd, lub sijhawm siv ultrasonic-pab tau muab piv rau ntawm 40 thiab 60 min, thiab 60 min ultrasonic da dej tsis muaj peev xwm muab rho tawm ntau dua 40 min; li no, 40 min tau teem caij rau ultrasonic apparatus rau lub sij hawm-saving xav.

Ntawm qhov tod tes, {{0}}.5 mL ntawm CD3OD-phosphate buffer hauv D2O (1: 1, pH 7.4) tau muab piv nrog 0.5 mL ntawm CD3OD-D2O (1: 1). Ib qho 2.0 mL aliquot ntawm cov extract tau ua tiav cov ntsiab lus, rov tsim dua tshiab, thiab raug rau NMR tsom xam kom them tau cov lus teb tsim nyog rau feem ntau cov cim. Tsis tas li ntawd, txoj kev ntub dej tau pom tias zoo dua li qhov kev pab cuam presaturation kom txo qis qhov siab tshaj plaws ntawm cov dej seem (nyob ib ncig ntawm δ 4.8) hauv spectrum. Peb kuj pom tias qhov nce ntawm tus lej scanning tau muaj txiaj ntsig zoo rau kev txhim kho cov teeb liab-suab nrov piv (S / N) uas muaj txiaj ntsig zoo rau kev tshawb pom rhiab heev (tshwj xeeb rau cov khoom ntawm cov kab ke). Txawm li cas los xij, txij li txoj hauv kev no ua rau muaj kev phom sij rau kev ntsuas nrawm, 256 qhov kev soj ntsuam tau siv thaum kawg rau txhua qhov 1H NMR kev soj ntsuam kom ua tiav qhov kev xav tau.

3.2. Kev teeb tsa ntawm 1H NMR spectrum

Kev txheeb xyuas cov tshuaj lom neeg hauv CD tau ua tiav los ntawm kev sib koom ua ke ntawm 1H NMR, 13C NMR, thiab 2D NMR spectra (Fig. 1 thiab Cov Ntaub Ntawv Ntxiv Fig. S1-S10) thiab muab piv nrog cov qauv ntawm cov ntsiab lus tseeb, nrog rau kev xa mus rau kev nkag mus tau. databases, xws li MMCD. Cov kev ua tau zoo ntawm cov teeb liab hauv 1H NMR tau muab rau hauv daim duab 1. Kev hloov pauv tshuaj lom neeg rau cov cim qhia tau raug sau tseg hauv Table S2 (cov ntaub ntawv ntxiv).

Cov yam ntxwv ntawm PhGs hauv genus Cistanche tau piav qhia hauv cov ntawv nyeem 1. Vim hais tias ntawm cov qauv zoo sib xws ntawm PhGs, piv txwv li, echinacoside vs. acteoside, proton signals ntawm phenylethanoid moieties feem ntau sib tshooj nyob rau hauv 1H NMR spectrum. Qhov kev sib tshooj no ua rau muaj teeb meem kev tshuaj ntsuam nyuaj kom ntseeg tau tias muaj kev ntxub ntxaug ntawm cov teeb liab no. Nyob rau hauv txoj kev tshawb no tam sim no, cov teeb liab uas yog echinacoside thiab acteoside, uas yog cov khoom xyaw tseem ceeb hauv cov nroj tsuag qub, tau muab tsis meej nrog kev pab cuam ntawm cov khoom siv sib xyaw thiab ntau hom NMR spectra (Table S2, Fig. 1 thiab Fig. S1-S6). Cov teeb liab kuaj mob ntawm δ 7.73 (d, J 16.0 Hz) qhia txog kev faib tawm Castano-

sab B/D lossis lwm yam feruloyl hloov PhGs (Table S2 thiab daim duab 1) 22. Ntxiv mus, cis-type PhGs (cis-type coumaroyl los yog caffeoyl hloov PhGs) kuj tau pom nyob rau hauv cov extract raws li muaj δ 6.95 (d, J 12.0 Hz) nyob rau hauv spectrum (Table S2 thiab Fig. . 1).

Ob peb lub cim pom tseeb tau kuaj pom hauv thaj tsam δ 8.0–9.5. Cov teeb liab ntawm δ 9.15, 8.88, thiab 8.09 tau muab tso rau nicotinamide (Table S2 thiab Fig. 1), thiab 13C NMR nrog rau 2D

NMR spectra (Fig. S4–S6) kuj txhawb txoj haujlwm no. Lub teeb liab ntawm δ 8.48 tau plausibly muab rau formic acid, whereas cov teeb liab ntawm δ 8.13 thiab 8.11 tau tsim los ntawm adenosine thiab adenine, raws li (Table S2 thiab Fig. 1). Cov teeb liab carbohydrate feem ntau nyob ntawm thaj tsam ntawm δ 3.00 thiab 5.50. Obviously, sucrose yog cov disaccharide ntau tshaj plaws hauv CD, nthuav tawm qhov tseem ceeb ntawm resonances ntawm δ 4.04, 4.18, thiab 5.41 (Table S2 thiab Fig. 1 thiab S7). Cov cim qhia pom tseeb ntawm cov anomeric protons ntawm -galactose, -glucose, thiab -glucose tau resonated ntawm δ 5.24, 5.20, thiab 4.60, raws li (Table S2, Fig. 1 thiab S8, S9). Qhov tshwm sim ntawm mannitol tau nthuav tawm los ntawm kev soj ntsuam ntawm lub teeb liab ntawm δ 4.00 nrog rau kev sib piv nrog cov ntaub ntawv siv (Table S2, Fig. 1 thiab S10). Cov teeb liab kuaj mob ntawm 1-O-ethyl-glucoside tau pom ntawm 1.16 ppm los ntawm kev xa mus rau HMDB. Amino acids, suav nrog leucine (0.93 ppm), isoleucine / valine (0.98 ppm), threonine (1.27 ppm), alanine (1.49 ppm), lysine (1.71 ppm), glutamine (2.30 ppm), asparagine acid (2.96 ppm), (4.09 ppm), tyrosine (7.12 ppm), thiab phenylalanine (7.33 ppm) tau txais kev tso cai los ntawm kev sib piv lawv cov kev kuaj mob spectroscopic nrog cov ntaub ntawv khaws tseg hauv HMDB (Table S2 thiab Fig. 1). Iridoids thiab lignans tau hais los saum no ua cov tshuaj lom neeg tseem ceeb hauv CD. Hauv tus neeg sawv cev 1 H NMR spectrum (Fig. 1), cov cim ntawm syringaresinol (ib lignan derivative), thiab 8-epiloganic acid (ib qho iridoid derivative) tau pom ntawm δ 7.02 thiab 0.98, raws li. Ntxiv mus, lub multiplet signals ranged ntawm δ 6.56–6.64 tuaj yeem raug muab tso rau citrus A, alaschanioside A, lossis dehydeodiconiferyl cawv glucoside (tag nrho cov lignan derivatives), thaum cov teeb liab ntawm δ 6.18–6.33 kuj tseem tuaj yeem tsim los ntawm iridoids thiab 22. Daim duab 1). Tsis tas li ntawd, qhov tshwm sim ntawm qee cov aliphatic carboxylic acids hauv CD, suav nrog fumaric acid, txiv neej acid, malic acid, isocitric acid, citric acid, ketoglutaric acid, pyruvic acid, succinic acid, acetic acid, thiab lwm yam fatty acids, yog tentatively characterized by the Kev soj ntsuam ntawm cov teeb meem kev kuaj mob ntawm δ 6.54, 6.02, 4.27, 3.04, 2.73, 2.52, 2.49, 2.42, 1.97, thiab 1.33, ua ntu zus (Table S2 thiab daim duab 1). Tsis tas li ntawd, qee cov teeb liab, xws li cov teeb liab sib txawv nyob ib puag ncig 3.80 ppm thiab ib lub singlet ntawm 1.97 ppm22, tau suav tias yog cov yam ntxwv rau methoxy thiab acetyl pawg (Table S2 thiab Fig. 1), feem, uas yog cov kev hloov pauv ntawm phenyl derivatives. , tshwj xeeb tshaj yog PhGs hauv qhov no. Lub caij no, lub teeb liab ntawm 1.06 ppm tuaj yeem raug xa mus rau rhamnose residue. Nws paub zoo tias 1 H NMR spectroscopy tuaj yeem muab cov ntaub ntawv ncaj qha rau ntau vim tias qhov kev siv ntawm cov teeb liab proton yog proportional rau molar concentration ntawm analyte19. Yog li ntawd, qhov kev sib piv ntawm qhov ua ntej tuaj yeem ua rau cov khoom xyaw hauv CD. Pom tseeb, cov carbohydrates, tshwj xeeb tshaj yog sucrose, tau txais cov lus teb siab tshaj plaws hauv cov neeg sawv cev spectrum (Daim duab 1). Raws li cov nroj tsuag holoparasitic uas loj hlob hauv av yuav luag tag nrho lub neej voj voog, photosynthesis tsis tsim nyog thiab tsis muaj rau CD; Yog li ntawd, nws tsis yog qhov xav tsis thoob tias tsis muaj cov txheej txheem photosynthesis cuam tshuam nrog cov metabolite, xws li Kelvin cycle (tseem hu ua C3 cycle) cov neeg koom, tau pom nyob rau hauv spectrum23. Ntawm qhov tsis sib xws, muaj txiaj ntsig ntawm cov molecules koom nrog lub voj voog tricarboxylic acid (TCA lub voj voog, tseem hu ua citric acid cycle thiab Krebs cycle) tau kuaj pom, xws li citric acid, ketoglutaric acid, pyruvic acid, thiab succinic acid, qhia tias muaj zog hauv nruab nrab. carbon metabolism (CCM) tshwm sim nyob rau hauv thawj cov nroj tsuag. Ntawm qhov tod tes, PhGs tau pom tias yog tsev neeg tshuaj lom neeg ntawm ntau yam metabolites theem nrab, thaum lignans thiab iridoids tsuas yog muab kev pab me me rau tag nrho cov spectral profile.

3.3. Precision assay ntawm 1H NMR spectroscopy

Lub spectroscopic precision plays lub luag haujlwm tseem ceeb hauv kev ntseeg siab ntawm metabolomics characterization. Nyob rau hauv txoj kev tshawb no tam sim no, qhov tseeb ntawm tag nrho cov txheej txheem tau soj ntsuam los ntawm kev npaj ib tus qauv sawv cev (CD1-GUI) hauv quintuplicate thiab tom qab ntawd tshuaj xyuas nws hauv ob hnub sib law liag. Cov tau txais spectra tau nthuav tawm qhov zoo sib xws los ntawm kev sib tshooj tag nrho cov spectra, qhia tias cov qauv kev npaj raws tu qauv thiab 1H NMR ntsuas tau rov tsim dua thiab cov qauv tuaj yeem ruaj khov hauv ob hnub tsawg kawg.

3.4. Kev ntxub ntxaug ntawm cov nroj tsuag sab saud thiab qis

Txog rau tam sim no, tsis muaj pov thawj pom tau tias qhov chaw sab saud ntawm CD yog qhov sib npaug ntawm qhov qis ntawm cov tshuaj profile thiab cov khoom siv tshuaj. Lub hom phiaj kom paub meej seb puas muaj qhov sib txuam ntawm thawj lossis theem nrab metabolites tshwm sim hauv qee qhov chaw ntawm CD, tag nrho cov succulent stems raug txiav mus rau sab sauv thiab qis, ob qho tib si tau ua tiav tom qab, muab rho tawm, thiab ntsuas siv 1 H NMR spectroscopy. Fig. 2 nthuav qhia tus neeg sawv cev ntawm ob qho tib si. Zuag qhia tag nrho, feem ntau cov cim qhia nyob rau hauv qis dua yog siab dua cov nyob rau sab sauv. Hauv qhov ntau ntawm δ 6.0-9.5, tsis muaj lub cim tshwj xeeb tuaj yeem pom nyob rau hauv ob qho tib si; Txawm li cas los xij, tag nrho cov kev siv ntawm cov teeb liab nyob rau hauv qis qis yog me ntsis siab dua cov nyob rau hauv lub sab sauv qhov chaw, qhia hais tias cov aromatic derivatives yuav enriched nyob rau hauv lub domain ze rau parasitic point23. Cov qis qis kuj tseem muaj cov roj fatty acids, qee cov amino acids, thiab qee cov khoom siv hluav taws xob vim tias tag nrho cov teeb liab teb ntawm thaj av aliphatic (δ 0.5-3.{10}} }) nyob rau hauv qis qis yog siab dua li ntawm cov sab sauv. Ntxiv mus, qee qhov sib txawv pom tseeb tuaj yeem pom hauv thaj tsam qab zib (δ 3.0-5.0), thiab feem ntau ntawm cov teeb liab nyob hauv thaj tsam ntawm δ 3.0- 5. Txhawm rau hais txog qhov sib txawv ntawm qhov nruab nrab ntawm qhov nruab nrab thiab qis qis thiab tseem txhawm rau txheeb xyuas cov neeg koom nrog hauv kev saib xyuas lawv qhov kev ntxub ntxaug, kev txheeb xyuas cov ntaub ntawv sib txawv tau siv los ua cov txheej txheem spectroscopic dataset ntawm qhov sib txawv. Ib txoj hauv kev uas tsis muaj kev saib xyuas npe hu ua PCA, uas tsuas yog siv cov ntaub ntawv los ntawm ib qho matrix, tau pib siv txhawm rau txheeb xyuas tag nrho cov qauv raws li tus yam ntxwv 1 H NMR spectra. Txawm li cas los xij, kev sib tshooj dav dav tshwm sim rau ob qhov sib txawv no (cov ntaub ntawv tsis qhia). Tom qab ntawd, OPLS-DA, ib txoj hauv kev saib xyuas, tau pib ua kom pom kev sib cais ntawm cov pab pawg sib txawv nrog rau kom nkag siab txog qhov sib txawv uas nqa cov ntaub ntawv sib cais hauv chav kawm. OPLS-DA tau ua tiav qhov kev sib cais zoo rau qhov sib txawv ntawm CD. Daim duab 3A thiab B qhia cov qhab nia thiab S-plot ntawm OPLS-DA, raws li. Ob qho tag nrho qhov zoo ntawm qhov haum (R2Y¼{{30}}}.910) thiab tag nrho qhov sib npaug ntawm qhov sib npaug (Q2Y¼0.981) tau ze rau 1.0. Yog li ntawd, tus qauv sib cais thawj zaug tau suav nrog lub suab nrog kev kwv yees siab. Pom tseeb, qhov chaw sab saud tuaj yeem sib txawv ntawm qhov qis dua thaum cov qauv tau sau nrog ob pawg, thiab S-plot muab cov cim qhia uas muaj feem cuam tshuam rau qhov sib txawv. Zuag qhia tag nrho, ntau cov dots tau muab faib rau hauv thaj av uas sib haum mus rau qhov qis hauv S-plot, qhov twg ntau dua cov ntsiab lus ntawm fatty acids (δ 1.32–1.35) thiab TCA cycle factor (xws li succinic acid ntawm δ 2.42) raug faib; Txawm li cas los xij, sab sauv (txoj cai hauv daim duab 3A) tau nplua nuj nyob hauv qee cov carbohydrates (tsuas yog δ 3.70–4.10, Fig. 3B). Lub peev xwm biomarkers muab los ntawm S-plot tau zoo sib xws nrog kev sib piv ntawm cov spectra ntawm ob ntu los ntawm kev soj ntsuam ncaj qha thiab overlaying. Nws txhais tau hais tias cov metabolites yog synthesized nyob rau hauv cov ntaub so ntswg-, lub cev thiab kev loj hlob-txheej txheem txoj kev los ntawm tej biosynthesis enzymes thiab ces muab cia, tej zaum nyob rau hauv cov ntsiab lus siab nyob rau hauv lub zus tau tej cov domains, sib haum mus rau lawv ntau yam functions rau tag nrho cov nroj tsuag. Piv txwv li, ob qho tib si ntawm hom thiab cov ntsiab lus ntawm ginsenosides nthuav tawm qhov txawv txav ntawm cov rhizomes, cov hauv paus hniav, nplooj, thiab cov neeg ntawm Panax notoginseng24. Hais txog CD, los ntawm qhov kev pom zoo, cov txheej txheem metabolite thib ob tau nthuav tawm qhov zoo sib xws ntawm sab sauv thiab sab qis. Vim tias qhov qis ntawm tag nrho cov nroj tsuag ua lub luag haujlwm rau kev xa cov khoom noj khoom haus, feem ntau yog cov metabolites thawj, piv txwv li, carbohydrates, los ntawm tus tswv tsev, Haloxylon ammodendron mus rau cov ntsiab lus tseem ceeb ntawm cov metabolism hauv lub cev, qhia tau hais tias ntau cov metabolites tseem ceeb yuav tsum tshwm sim hauv lub cev. qis dua. Ntxiv mus, nws kim heev hydrolysis ntawm polysaccharides mus rau oligosaccharides thiab tom qab ntawd mus rau monosaccharides, uas yuav tsum tau bio-hloov mus rau hauv ib co aliphatic carboxylic acids, yuav tsum tau tshwm sim nyob rau hauv lub Upper qhov chaw. Vim tias 50 feem pua ​​​​aqueous methanol tau siv rau kev rho tawm hauv txoj kev tshawb no thiab nws muaj peev xwm rho tawm hydrophilic tsis tshua muaj molecules es tsis txhob ntawm macromolecules (xws li polysaccharides), nws tsis yog qhov xav tsis thoob tias qhov sib xyaw ntawm TCA lub voj voog thiab cov roj fatty acids tau pom nyob rau hauv qis dua. ntu. Txawm li cas los xij, qee qhov oligosaccharides, cov khoom siv hydrolytic ntawm polysaccharides, tau pom tias muaj kev nplua nuj nyob rau sab saud. Ntawm qhov tod tes, qhov sib txawv me me tau pom zoo rau PhGs ntawm qhov sib txawv los ntawm qhov kev xav ntau, thiab tag nrho, qhov me me ntawm PhGs tau pom nyob rau hauv qis dua 23,25. Nws tau tshaj tawm tias haustorium phloem yog tej zaum cov khoom siv hluavtaws thib ob rau PhGs hauv CD23; yog li ntawd, qhov qis dua, uas nyob ze rau qhov parasitic point, yuav tsum muaj nyiaj PhGs.

3.5. Kev pom zoo ntawm txoj kev ua haujlwm tshiab rau CD

Obviously, cov tshuaj pleev xim rau cov qauv c-hom tau zoo tshaj plaws ntawm cov tsos zoo, xim dawb, lub teeb, thiab kev ntxhib los mos. Daim duab 4 qhia tus neeg sawv cev 1 H NMR spectra rau peb hom qauv. Tom qab kev ntsuas sib npaug, txawm li cas los xij, sib txawv spectral profiles tau txais rau cov txheej txheem tshuaj. Los ntawm kev soj ntsuam ncaj qha thiab overlaying, pom tseeb qhov sib txawv tuaj yeem pom. Ua ntej, nyob rau hauv cheeb tsam ntawm δ 6.00-8.00, uas feem ntau suav nrog cov cim qhia txog PhGs thiab qee qhov lwm yam phenolic derivatives, cov lus teb ntawm c-hom qauv (Fig. 4C) yog heev dua lwm yam ob hom. Qhov thib ob, cov lus teb ntawm feem ntau cov carbohydrates thiab TCA cov neeg koom hauv cov qauv b (Daim duab 4B) yuav luag sib npaug rau cov hauv cov qauv c (daim duab 4C) tab sis ntau dua li cov qauv hauv ib hom (Fig. 4A) . Thib peb, cov ntsiab lus ntawm cov piam thaj (δ 5.20 rau -glucose, thiab δ 4.60 rau -glucose) hauv cov qauv b (daim duab 4B) yog me ntsis siab dua li cov qauv c-hom (Fig. 4C). Thaum kawg, kuaj pom cov roj fatty acid ntau dua hauv cov qauv a-thiab c-types dua li cov qauv b (Fig. 4). Qhov tseem ceeb tshaj plaws, ntau tshaj phenol derivatives, xws li qhov no, uas tau pom dav dav raws li cov khoom xyaw zoo hauv CD, tau pom nyob rau hauv cov qauv ua tiav los ntawm ib txoj kev tshiab (c-hom qauv), hauv kev sib piv ntawm a-thiab b- hom qauv. Tsis tas li ntawd, ntau cov metabolites tseem ceeb, xws li carbohydrates, cov neeg koom nrog TCA, thiab fatty acids kuj pom nyob rau hauv cov qauv c. Tom qab ntawd, kev sib piv ntawm tag nrho cov qauv los ntawm ntau qhov sib txawv ntawm cov ntaub ntawv tshuaj ntsuam tau ua los txhawm rau txhawm rau hais txog qhov zoo sib xws nrog rau qhov sib txawv ntawm peb hom tshuaj ntsuab. PCA nrog Par scaling thiab OPLS-DA nrog UV scaling tau ua los ua cov ntaub ntawv NMR spectroscopic, ua tiav (cov ntaub ntawv tsis qhia). Kev sib cais tseem ceeb tsis tau tiav siv PCA; Txawm li cas los xij, cov qhab nia ntawm OPLS-DA rau peb pawg no (ntsuab, xiav, thiab liab dots hauv daim duab 5A) tau qhia meej kev faib tawm, qhia tias peb pawg qauv no txawv heev txog lawv cov metabolic profiles. Cov

degradation ntawm PhGs pib los ntawm cov enzymes nyob rau hauv thawj cov nroj tsuag los yog high-temperature cia. Raws li qhov tshwm sim, cov txheej txheem ua tiav (c-hom qauv) siv txoj kev tshiab tau zoo ntawm ob qho tib si zoo nkauj thiab zoo dua cov ntsiab lus sib xyaw. Txhawm rau txheeb xyuas cov txiaj ntsig tau hais los saum toj no rau peb txoj kev ua tiav, HPLC-UV txoj kev uas tau tsim zoo thiab muaj pov thawj hauv Suav Pharmacopeia (2015 tsab) 3 tau siv los txiav txim siab cov ntsiab lus ntawm echinacoside thiab acteoside, uas ua lub luag haujlwm tseem ceeb rau paub qhov txawv ntawm peb hom khoom ua tiav saum toj no. Cov ntsiab lus nruab nrab ntawm echinacoside hauv cov qauv a-, b-, thiab c-hom qauv yog 8.33, 4.10, thiab 16.19 mg / g, raws li, thaum cov ntsiab lus ntawm cov acteoside yog 1.57, 1.14, thiab 3.66 mg / g, feem. Pom tseeb, PhGs cov ntsiab lus hauv cov ntaub ntawv c-hom yog 2-4 folds siab dua li cov qauv hauv a-thiab b-hom qauv, pom zoo nrog 1 H NMR-based metabolomics cov txiaj ntsig. Ntawm cov txheej txheem tshuaj ntsuam xyuas muaj feem ntau ua haujlwm hauv kev tshawb fawb metabolomics, NMR thiab MS-raws li txoj hauv kev tau lees paub tias yog lwm txoj hauv kev zoo. NMR spectroscopy, tshwj xeeb tshaj yog 1 H NMR, muaj qhov tsis muaj txiaj ntsig zoo dua li lwm cov tswv yim, xws li kev xaiv tsis yog, kev npaj ua qauv yooj yim, thiab qhov yooj yim ntawm kev tshawb nrhiav ib txhij ntawm ntau pawg ntawm metabolites hauv lub sijhawm ntsuas luv luv. Txog tam sim no, 1 H NMR-raws li metabolomics tau siv los txheeb xyuas cov nroj tsuag17 los sib piv cov tshuaj analogue18, kom sib txawv qhov chaw nyob 26, thiab ua kom pom qhov degradation ntawm tshuaj ntsuab26. Ntawm no, 1 H NMR tau siv los paub qhov txawv ntawm qhov sib txawv ntawm CD, thiab tseem yuav nthuav tawm cov txheej txheem ua haujlwm tshiab, qhia tias 1 H NMR spectroscopy yog qhov hloov pauv tau thiab muaj zog analytical cuab yeej los muab cov lus qhia tseem ceeb rau kev siv CD thiab lwm yam. tshuaj ntsuab los ntawm kev muab cov ntaub ntawv zoo thiab ntau cov ntaub ntawv ntawm cov nyom extract matrices.