Dereplication-Guided Isolation Of New Phenylpropanoid-Supstituted Diglycosides From Cistanche Salsa Thiab Lawv Inhibitory Activity On NO Production in Macrophage

Thov hu rauoscar.xiao@wecistanche.comrau ntau

Abstract:

Dereplication tso cai rau kev txheeb xyuas sai ntawm cov paub thiab tsis paub cov tebchaw hauv cov nroj tsuag rho tawm. Hauv txoj kev tshawb no, peb tau ua kua chromatography-mass spectroscopy (LC-MS)-raws li dereplication siv cov ntaub ntawv los ntawm ESI ntxiv rau QTOF-MS rau kev tsom xam ntawmphenylpropanoid hloov glycosides, cov koom haum tseem ceeb ntawm Cistanche salsa (CA Mey.) Beck. Siv TOF-MS ib leeg, cov txheej txheem ntawm cov khoom sib txuas no tuaj yeem lees paub tsis meej raws li tus yam ntxwv fragmentation qauv ntawm ntau yam kev tsim khoom. HPLC-MS-raws li profileing ntawm C. salsa kuj tso cai rau kev tshawb nrhiav cov tshiabphenylpropanoid tshuaj- hloovglycosides los ntawmtsob nroj no. Ntawm lawv, tsib yam tshiab phenylpropanoid hloov pauvdiglycosides, lub npe cistansalsides A-E (5, 6, 12, 17 thiab 18), raug cais tawm. Lawv cov qauv tau piav qhia los ntawm cov txheej txheem spectroscopic suav nrog NMRand MS tsom xam. Tag nrho cov kev sib cais tau raug sim rau lawv cov kev tiv thaiv kev tiv thaiv tsis muaj kev tsim tawm hauv RAW 264.7 hlwb txhawb nqa los ntawm LPS. Ntawm cov tshuaj ntsuam xyuas, cov tebchaw 5, 11, 13 thiab 18 tau pom tias muaj kev cuam tshuam me ntsis ntawm kev ua haujlwm tsis muaj zog ntawm NO synthase. Cov tshuaj 11, 13 thiab 18 kuj inhibited phosphorylation ntawm NF-κB hauv macrophages. Tsis muaj qhov sib xyaw ua ke pom qhov tseem ceebcytotoxicity. 

Ntsiab lus:dereplication; phenylpropanoid-hloov diglycosides; Cistanche salsa; anti-inflammatory

Thov nias ntawm no kom paub ntxiv

Taw qhia

Cistanche salsa (CA Mey.) Beck, belongs rau tsev neeg Orobanchaceae, yog cov kab mob cab uas tau txais nws cov khoom noj los ntawm cov hauv paus hniav ntawm Haloxylon ammodendron (Chenopodiaceae) thiab lwm cov nroj tsuag suab puam [1]. Cov nroj tsuag no tau siv rau hauv cov tshuaj ib txwm siv rau kev kho mob ntawm neurasthenia, kev sib deev dysfunction thiab raum tsis muaj peev xwm [2,3]. Hauv kev tshawb fawb phytochemical yav dhau los, nws tau raug tshaj tawm tias tag nrho cov nroj tsuag ntawm C. salsa muaj ntau hom tshuaj xws li phenylethanoid glycosides thiab iridoid glycosides [4–7]. Phenylethanoid glycosides, xws li acteoside thiab echinacoside, yog cov khoom tseem ceeb ntawm cov nroj tsuag [8]. Cov extracts ntawm C. salsa pom cov khoom muaj txiaj ntsig, suav nrog kev tiv thaiv kab mob, tshuaj tiv thaiv kab mob thiab tiv thaiv kev ua haujlwm [9,10]. Dereplication yog ib qho txheej txheem uas cov qauv sib xyaw ua ke yuav raug kuaj kom paub qhov txawv ntawm cov khoom tsis paub los ntawm cov sib txuas paub. Cov tswv yim dereplication yog ua raws li cov txheej txheem kev tshuaj xyuas thiab kev tshawb nrhiav cov ntaub ntawv los txheeb xyuas cov metabolites theem nrab thaum ntxov hauv cov txheej txheem tshawb fawb phytochemical [11]. Ntawm cov txheej txheem analytical, ESI-QTOF-MS (electrospray ionization-quadrupole-lub sij hawm ntawm davhlau-mass spectroscopy) tuaj yeem muab cov ntaub ntawv tseem ceeb txog cov qauv tshuaj ntawm cov metabolites theem nrab. Lub LC-MS-raws li dereplication-guided fractionation tau pom tias yuav pab kom extraction thiab purification ntawm lub hom phiaj metabolites los ntawm crude extracts ntawm cov nroj tsuag nrog high efficiency [12-15]. Txoj kev tshawb no tau ua LC-MS-based dereplication siv cov ntaub ntawv los ntawm ESI ntxiv rau TOF-MS rau kev tshuaj xyuas ntawm phenylpropanoid-hloov diglycosides, cov khoom tseem ceeb ntawm C. salsa. Cov ntaub ntawv TOF-MS tuaj yeem hais qhia cov qauv ntawm cov khoom sib txuas no raws li tus yam ntxwv fragmentation qauv ntawm ntau yam khoom ions. Raws li qhov dereplication no, LC-MS profiles ntawm ethyl acetate (EtOAc) feem thiab dej-soluble feem raug tshuaj xyuas. Qhov feem EtOAc tau raug rau lub tswv yim dereplication rau kev sib cais ntxiv, ua rau muaj kev sib cais ntawm tsib lub tshiab phenylpropanoid-hloov diglycosides thiab 13 paub cov tebchaw (Daim duab 1). Nws tau raug lees paub tias qhov kev kwv yees tau kwv yees cov qauv ntawm phenylpropanoid-hloov cov diglycosides tau raug sib raug zoo rau lawv cov qauv tiag. Tsis tas li ntawd, cov tshuaj tiv thaiv kab mob ntawm cov kev sib cais tau tshawb pom.

Cov txiaj ntsig thiab kev sib tham

Cov phenylpropanoid-hloov diglycosides cais los ntawm C. salsa feem ntau muaj cov qauv raws li disaccharide glycosides, uas muaj cov piam thaj thiab cov rhamnose nrog Rha (1 → 3) Glc linkage thiab ib qho cinnamoyl hloov pauv, xws li coumaric acid (Cou), caffeic. acid (Caf) thiab feruloyl acid (Fer), ntawm C-4 lossis C-6 txoj haujlwm ntawm qabzib. Lub aglycone feem ntau txuas rau ntawm C-1 txoj haujlwm ntawm qabzib. Cov qauv ntawm phenylpropanoid-hloov diglycosides nrog ib pawg acetyl ntawm C-2 ntawm cov piam thaj tau nquag tshaj tawm [6,12,16]. Txhawm rau ua qhov dereplication, MS fragmentation qauv ntawm cov tebchaw no tau txheeb xyuas los ntawm hom zoo ESI-QTOF-MS. Hauv MS spectra, tag nrho cov phenylpropanoid-hloov cov diglycosides tsim cov khoom siv ion peaks ntawm [M ntxiv rau NH4] ntxiv , [M ntxiv rau K] ntxiv thiab [M ntxiv Na] ntxiv, uas muab cov khoom hnyav thiab cov mis. Cov qauv ntawm fragment ions tuaj yeem pom los ntawm kev poob ntawm aglycone thiab glycoside residues ([M ntxiv rau H − Aglycone] ntxiv , [M ntxiv H − Aglycone − Rha] ntxiv thiab [M ntxiv H − Aglycone - Daim duab 1. Cov qauv ntawm cov tebchaw 1–18. 2. Cov txiaj ntsig thiab Kev Sib Tham Cov phenylpropanoid-hloov cov diglycosides cais los ntawm C. salsa feem ntau muaj cov qauv raws li disaccharide glycosides, uas muaj cov piam thaj thiab cov rhamnose nrog Rha (1→3) Glc linkage thiab ib qho cinnamoyl hloov, xws li coumaric acid (Cou), caffeic acid (Caf) thiab feruloyl acid (Fer), ntawm C-4 lossis C-6 txoj haujlwm ntawm qabzib. Cov aglycone feem ntau txuas ntawm C{{24. Cov qauv ntawm phenylpropanoid-hloov diglycosides nrog ib pawg acetyl ntawm C-2 cov piam thaj tau nquag tshaj tawm [6,12,16]. Txhawm rau ua qhov dereplication, MS fragmentation qauv ntawm cov tshuaj no. Hauv MS spectra, tag nrho cov phenylpropanoid-hloov diglyco sab tsim adduct ion peaks ntawm [M ntxiv rau NH4] ntxiv , [M ntxiv rau K] ntxiv thiab [M ntxiv Na] ntxiv, uas muab qhov hnyav molecular thiab mis. Cov qauv ntawm fragment ions tuaj yeem pom los ntawm kev poob ntawm aglycone thiab glycoside residues ([M ntxiv H − Aglycone] ntxiv , [M ntxiv H − Aglycone − Rha] ntxiv thiab [M ntxiv H − Aglycone - Rha − Glc (los yog Acetyl -Glc)] ntxiv rau), uas yog qhov tseem ceeb rau kev kwv yees hom cinnamoyl hloov pauv thiab qab zib. Cov fragment ions ntawm m / z 163 ntawm pawg caffeoyl, m / z 147 ntawm pawg coumaroyl lossis m / z 177 ntawm pawg feruloyl muab cov yam ntxwv teeb liab ntawm cinnamoyl hloov pauv hauv phenylpropanoid-hloov diglycosides [4,15] ( Daim duab 2). Kev tsom xam ntawm cov fragment ions yuav muab cov ntaub ntawv tseem ceeb rau kev txheeb xyuas cov qauv ntawm phenylpropanoid-hloov diglycosides. Txawm li cas los xij, lawv cov isomers tsis tuaj yeem sib txawv los ntawm MS spectrometry ib leeg. Txhawm rau txheeb xyuas qhov tseeb ntawm lawv cov qauv ua tiav, NMR spectra yuav tsum tau ua.

Tag nrho cov kev sib cais tau raug sim rau lawv cov teebmeem inhibitory ntawm LPS-vim tsis muaj ntau lawm hauv RAW 264.7 hlwb. Dexamethasone tau siv los ua kev tswj hwm zoo thiab nws cov IC50 yog 7.0 µM. Ntawm cov tshuaj ntsuam xyuas, cov tebchaw 5 (IC50 42.7 ± 6.6 µM), 11 (IC50 37.3 ± 2.2 µM), 13 (IC50 40}{2{{ 24}}}} ± 4. tus nqi> 100 µM). Txhawm rau txheeb xyuas seb cov tshuaj no puas muaj cytotoxicity, cell viability raug ntsuas los ntawm kev siv MTT kev soj ntsuam. Yog li ntawd, tsis muaj leej twg ntawm lawv tau nthuav tawm cytotoxicity tseem ceeb (Cov Duab Ntxiv S6-1). Cov plaub lub tebchaw no tau raug xaiv los ntsuas rau lawv cov haujlwm inhibitory tiv thaiv NF-κB txoj hauv kev LPS-stimulated RAW 264.7 hlwb. Stimulation ntawm RAW 264.7 hlwb nrog LPS induced phosphorylation ntawm IκB thiab NF-κB (p65) tom qab 0.5 h ntawm incubation. Lub phosphorylation ntawm NF-κB (p65) tau txo qis los ntawm pretreatment nrog cov tebchaw 11, 13 thiab 18 raws li qhia los ntawm western blot tsom (Daim duab 5). Yog li, cov tebchaw 11, 13 thiab 18 tuaj yeem ua rau muaj kev cuam tshuam los ntawm kev cuam tshuam ntawm NF-κB hauv macrophages.

Cov ntaub ntawv thiab cov txheej txheem

Kev sib hloov kho qhov muag tau ntsuas nrog Jasco P-2000 digital polarimeter (Jasco, Tokyo, Nyiv). UV spectra tau kaw rau ntawm Chirascan ntxiv rau Circular Dichroism spectrometer (Chirascan, APL, UK). IR spectra raug kaw siv Jasco FT/IR-4200 spectrophotometer. High-resolution electrospray ionization quadrupole-time-of-flight mass spectrometry (HR-ESI-qTOF-MS) tau ua ntawm Agilent 6530 Accurate-Mass Q-TOF LC/MS nruab nrog Agilent 1260 Infinity series (Agilent Technologies, Inc. , Palo Alto, CA, USA), thiab kem siv yog Jasco SFCpak Crest C18T-5 kem (id150 × 4.6 mm,5 um). MassHunter Workstation Software tau siv rau kev nrhiav tau cov ntaub ntawv.

1D (H thiab 13C) thiab 2D('H-1H COSY, HSQC, HMBC, NOESY) NMR spectra tau txais nrog Jeol LA 300 (Jeol, Tokyo, Nyiv), Bruker AVANCE-400, Bruker AVANCE-500, Bruker AVANCE-600 thiab Bruker AVANCE 800 HDspectrometers ua ke nrog cryoprobe (Bruker, Ettlingen, Lub teb chaws Yelemees). DMSO-d6 (Cambridge Isotope Laboratories, Inc. Andover, MA, USA) tau siv los ua NMR hnyav thiab siv peaks (6H 2.50 thiab δc 39.5). Kem chromatography (CC) tau ua tiav siv Sephadex LH-20(25-100 um; Pharmacia, Uppsala, Sweden) lossis Kieselgel 60 silica gel (40-63 um, 230-400 mesh, Art . 9385; Merck, Darmstadt, Lub teb chaws Yelemees). Nyias-txheej chromatography (TLC) tau ua nyob rau hauv pre-coated Kieselgel 60 silica gel F254 daim hlau (Art.5715; Merck).Spots ntawm TLCwere kuaj pom siv UVlamp ntawm 254 nm thiab 365 nm (VL{29}}.LC, 365 /254; Vilber Lourmat, Torcy, Fabkis). Medium pressure kua chromatography (MPLC) tau ua nyob rau ntawm RediSep 120g silica flash kem (lsco, Lincoln, NE, USA) thiab Kiesegel 60 silica gel (40-63 um,230-400 mesh, Art.9385; Merck ) siv tus khub Combiflash (Isco). Kev kub siab ua kua chromatography (HPLC) system yog Gilson HPLC nruab nrog Gilson 321 twj tso kua mis thiab UV.VIS 151 ntes (Gilson, Middleton, WI, USA), siv ib nrab npaj ODS txhua (Luna 5 um C18 (2) 100 Å, id 250 × 10 mm, 5 μm, Phenomenex Inc, Torrance, CA, USA; Hypersil GOLDrM aQ 175A,id250 × 10mm, 5 um, Thermo ScientificTM, Hennigsdorf, Lub teb chaws Yelemees; Inno C18column 120 A, id250 × 10 mm, 5 um, Young Jin Biochrom Co., Ltd., Seongana, Kauslim Teb). HPLC system yog Waters 2695 alliance system nrog 996 Photodiode Array (PDA) detector (Waters Corp, Milford, MA, USA), thiab kem siv yog HypersilTM BDS C18column (130 A, id150 × 4.6 mm, 5 μm, Thermo Scientific TM). Formic acid yog yuav los ntawm Daejung Chemicals & Metals Co., Ltd.(Seoul, Kauslim). HPLC qib cov kuab tshuaj tau yuav los ntawm Fisher Scientific Korea Ltd.(Seoul, Kauslim). HSOA, Na, CO, thiab cov kuab tshuaj qib thawj rau kev rho tawm, faib thiab cais tawm tau yuav los ntawm Daejung Chemical & Metals Co. Ltd. (Seoul, Kauslim). L- thiab D-cysteine ​​methyl ester hydrochloride thiab o-tolylisothiocvanate tau yuav los ntawm Tokyo Chemical Industry (Tokyo, Nyiv).

Cov lus xaus

Nyob rau hauv txoj kev tshawb no, peb cais thiab elucidated cov qauv ntawm tsib tshiab phenylpropanoid-hloov diglycosides, hu ua cistansalside AE ​​(5,6, 12, 17 thiab 18), ntxiv rau kev cais thiab txheeb xyuas 13 lub tebchaw paub, siv lub tswv yim dereplication. Cov tebchaw paub tau txiav txim siab tias yog lipedoside AI(1)[21], 2'-acetylacteoside (2)[22], isocistanoside C(3)[15], osmanthuside B (4)[21], epimeridinoside A(7) [15], cistanoside D(8)[23], salsaside B(9) [6l, tubuloside E (10)[16]cistanoside M(11)[15], isomartynoside(13) [24], salsaside C(14] )[6], jionoside C(15)[25] and salsaside F (16)[6]. Lawv cov qauv tau tsim los ntawm kev txheeb xyuas cov ntaub ntawv dav dav spectroscopic thiab los ntawm kev sib piv rau cov ntaub ntawv tshaj tawm hauv cov ntaub ntawv. Nws tau raug lees paub tias qhov kev kwv yees tau kwv yees cov qauv ntawm phenylpropanoid-hloov cov diglycosides tau raug sib raug zoo rau lawv cov qauv tiag.

Nyob rau hauv NO inhibitory assay, compounds 5 (IC50 42.7±6.6 μM), 11 (IC50 37.3±2.2 μM), 13 (IC50 40.0 ± 4. Ntawm cov tebchaw no, cov tebchaw 11,13 thiab 18 tau pom tias inhibit qhov phosphorylation ntawm NF-kB hauv macrophages thiab yog li yuav ua rau muaj kev tiv thaiv kab mob uas yuav tsum tau ua pov thawj hauv kev sim ntxiv.

Extracting los ntawm: https://creativecommons.org/licenses/by/4.0/ ("License"). Txawm hais tias ProQuest Terms and Conditions, koj tuaj yeem siv cov ntsiab lus no raws li cov ntsiab lus ntawm Daim Ntawv Tso Cai.