Kev npaj cais tawm thiab ua kom huv ntawm Plaub Lub Cheeb Tsam Los Ntawm Cistanches Deserticola YC Ma Los Ntawm Kev Kub Ceev Counter-Tam sim no Chromatography
Mar 06, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Abstract:
Ua raws li cov kauj ruam ntxiv ntawm cov silica gel, plaubphenylethanoid glycosidestau ntse cais los ntawmCistanches deserticolathiab purified los ntawm kev npaj high-speed counter-tam sim no chromatography (HSCCC) nrog ib tug ob-theem hnyav system muaj li ntawm ethyl acetate-n-butanol-ethanol-dej (40: 6: 6: 50, v/v /v/v). Tag nrho ntawm 30.9 mg acteoside, 13.0 mg isoacteoside, 12.5 mg syringalide A 3'- -L-rhamnopyranoside, thiab 7.2 mg 2'-acetylacteoside nrog purity siab dua 95 feem pua, raws li kev txiav txim los ntawm HPLC-ELSD, tau nyob rau hauv ib kauj ruam sib cais los ntawm 297 mg ntawmCistanche deserticola extract, raws. Lawv cov qauv raug txheeb xyuas los ntawm HR-MS, 1H-NMR, thiab 13C-NMR.
Ntsiab lus: high-speed counter-tam sim no chromatography;Cistanches deserticola YC Ma;phenylethanoid glycosides; acteoside; isoacteoside; syringalide A 3'- -L-rhamnopyranoside; 2'-acetylacteoside.
Taw qhia
Cistanches deserticolaYC Ma, a species ofCistanchesuas belongs rau Orobanchaceaefamily, yog ib tug paub zoo tshuaj suav tshuaj rau kev kho mob ntawm lub raum tsis muaj zog, poj niam infertility, morbid leucorrhea, neurapraxia, thiab senile cem quav [1]. Nws yog cov kab mob cab uas tau nthuav dav nyob rau sab qaum teb sab hnub poob ntawm Tuam Tshoj. Txog tam sim no, ntau lub tebchaw, suav nrog phenylethanoid glycosides (PhGs), iridoids, thiab lignans tau raug cais tawm ntawm hom no [2]. Qee qhov PhGs tau tshaj tawm tias muaj cov tshuaj tiv thaiv oxidative, hepatoprotective, thiab neuroprotective kev ua ub no [3–6]. Txawm li cas los xij, cov txheej txheem cais tawm thiab ua kom huv ntawm PhGs yog qhov nyuaj thiab siv sijhawm ntev. Ntxiv mus, classical ntau yam chromatographic txoj kev tsis tsuas yog siv ntau cov organic solvents tab sis kuj muab cov qauv qis dua. High-speed counter-tam sim no chromatography (HSCCC), ib qho kev txhawb nqa-dawb kua-kua muab faib chromatographic txheej txheem, muaj cov qauv zoo rov qab los piv rau qee cov qauv siv thiab tau siv dav rau kev sib cais thiab kev ua kom huv ntawm ntau yam khoom ntuj tsim [7–9] . Txawm hais tias ntau PhGs tau raug ntxuav los ntawmCistanchesgenus thiab lwm tus los ntawm HSCCC [10–14], tsis muaj ntawv qhia txog kev ua kom huv ntawm acteoside, isoacteoside, syringalide A 3'- -L-rhamnopyranoside thiab 2'-acetylacteoside hauv ib lub cev biphasic.
Hauv daim ntawv no, peb tshaj tawm ib txoj hauv kev yooj yim rau kev sib cais thiab ua kom huv ntawm plaub PhGs los ntawm C. deserticola. Thaum kawg, 30.9 mg ntawm acteoside, 13.0 mg ntawm isoacteoside, 12.5 mg ntawm syringalide A 3'- -L-rhamnopyranoside, thiab 7.2 mg ntawm 2'-acetylacteoside tau txais hauv ib kauj ruam. Sib cais los ntawm 297 mg ntawm feem nrog purities ntawm 99 feem pua, 95 feem pua, 99 feem pua, thiab 98 feem pua, feem. Lawv cov qauv tau tsim raws li 1H- thiab 13C-NMR spectral cov ntaub ntawv thiab HR-MS spectra. Cov qauv ntawm plaub PhGs tau txheeb xyuas hauv qhov kev tshawb nrhiav no tau qhia hauv daim duab 1.
Cov txiaj ntsig thiab kev sib tham
HPLC Kev Ntsuam Xyuas ntawm Cov Crude Extract
Cov ntsiab lus enriched feem ntawm n-butanoic extract los ntawm C. deserticola tau soj ntsuam los ntawm HPLC-UV. Cov kem siv yog Accurasil C18 (250 mm × 4.6 mm, id 5 μm) (Thermo-Fisher Scientific Instruments, nroog, xeev abbrev, USA), theem mobile yog methanol-dej (30:70, v/v). Tus nqi ntws yog 1 mL / min. Lub ntes wavelength yog teem rau ntawm 254 nm. HPLC chromatogram yog qhia hauv daim duab 2. Peaks 1 txog 4 sib raug rau acteoside (1), isoacteoside (2), syringalide A 3'- -L-rhamnopyranoside (3), thiab 2'-acetylacteoside (4), raws.
Kev xaiv ob-theem Solvent System
Kev sib cais ua tiav los ntawm HSCCC feem ntau yog nyob ntawm kev xaiv ntawm qhov tsim nyog ob-theem hnyav, ob-theem hnyav system raug xaiv raws li kev faib coefficient qhov tseem ceeb (K) ntawm txhua lub hom phiaj thiab qhov zoo tshaj plaws ntawm K yuav tsum yog los ntawm {{ 2}.5 rau 2.
Hauv kev sim, K qhov tseem ceeb rau lub hom phiaj ntawm ob peb lub cev hnyav tau pom nyob rau hauv Table 1 (txawm tias me ntsis siab dua rau qhov siab tshaj 4, cov txiaj ntsig tau pom tias ua tau). Ntawm lawv, ethyl acetate-n-butanol-ethanol-dej (40: 6: 6: 50, v/v/v/v) tau muab faib ua coefficients rau cov hom phiaj sib txuas. Thaum kawg, cov kuab tshuaj uas muaj li ntawm ethyl acetate-n-butanol- ethanol-dej (40: 6: 6: 50, v/v/v/v) tau siv los cais thiab ntxuav plaub lub tebchaw ntawm C. deserticola, raws li qhia hauv Daim duab 3. Raws li pom hauv daim duab 2, HPLC kev tshuaj xyuas ntawm txhua HSCCC feem qhia tau tias plaub cov PhGs ntshiab (acteoside, 99 feem pua; isoacteoside, 95 feem pua; syringalide A 3′- -L-rhamnopyranoside 99 feem pua thiab 2′- acetylacteoside 98 feem pua ) tuaj yeem tau txais los ntawm cov feem crude.
Kev sim
Cov cuab yeej
Cov khoom siv HSCCC ua haujlwm hauv txoj kev tshawb fawb tam sim no yog TBE-300B high-speed counter-tam sim no chromatography system (Shanghai Tauto Biotech Co., Shanghai, Suav teb) nrog peb txheej txheej txheej txheej txheej sib cais sib txuas hauv series (inch ntawm raj=2.6 hli, tag nrho ntim=300 mL) thiab 20 mL qauv voj. Lub vojvoog kiv puag ncig lossis qhov kev ncua deb ntawm tus tuav axis thiab nruab nrabaxis ntawm lub centrifuge (axis ntawm lub centrifuge (R) yog 5 cm, thiab tus nqi txawv ntawm 0.5 ntawm lub internal davhlau ya nyob twg mus rau 0.8 ntawm lub sab nraud davhlau ya nyob twg (= r/R, qhov twg r yog qhov kev ncua deb ntawm lub kauj mus rau tus tuav ncej). Ib qho HX 105 qhov ntsuas kub tsis tu ncua (Beijing Changliu Lab Instrument Company, Beijing, Suav) tau siv los tswj qhov kub thiab txias hauv qhov kev sim. Lub HSCCC system tau nruab nrog tus qauv TBP-5002 nruab nrab-siab qhov twj tso kua mis, tus qauv TBP-2000 UV ntes ua haujlwm ntawm 254 nm, thiab qauv V4.0 chaw ua haujlwm (Jinda Biochemistry Instrument Company, Shanghai, Suav teb).
Cov cuab yeej ua haujlwm siab ua kua chromatography (HPLC) siv yog Agilent 1200 system uas muaj G1312A binary twj tso kua mis, G1329A autosampler, G1314A qhov sib txawv wavelength detector (VWD), G1316A thermostatted kem compartment, a G1379 chaw ua haujlwm. Ib qho Alltech 3300 ELSD tau siv los kuaj xyuas cov purities. Agilent 6520 Q-TOF thiab Bruker AVANCE III 500-NMR spectrometers tau siv rau kev txheeb xyuas cov qauv sib xyaw. 1H thiab 13C-NMR spectra (ntawm 500 thiab 125 MHz, ntsig txog) tau ntsuas ntawm chav tsev kub (22 degree / 295.1 K).
Reagents thiab khoom siv
Ethyl acetate, n-butanol, thiab ethanol yog analytical qib thiab methanol yog qib HPLC. Tag nrho cov kuab tshuaj tau yuav los ntawm Tianjin Concord Technology Company (Tianjin, Suav). Milli-Q dej (18.2 MΩ) (Millipore, Bedford, MA, USA) yog siv rau tag nrho cov kev daws teeb meem thiab dilutions. Lub rhizome ntawm C. deserticola tau sau los ntawm Inner Mongolia Province, Tib neeg lub koom pheej ntawm Tuam Tshoj, thaum Lub Rau Hli 2009. Cov nroj tsuag tau txheeb xyuas los ntawm Prof. Lijuan Zhang, thiab daim ntawv pov thawj (No. 20091001) tau muab tso rau hauv peb lub chaw kuaj mob.
Kev npaj ntawm Crude Sample
Powdered air-dried fleshy stems of C. deserticola (1 Kg) tau refluxed ob zaug nrog aqueous ethanol (8 L, 60 feem pua v/v) rau 2 h txhua zaus. Cov extract tau evaporated nyob rau hauv txo siab thiab kub ntawm 60 degree kom txog thaum tag nrho evaporation ntawm ethanol. Cov residue raug tshem tawm hauv dej thiab muab rho tawm ua tiav peb zaug nrog chloroform, ethyl acetate, thiab n-butanol, them 5.16 g ntawm n-butanoic extract tom qab evaporation kom qhuav nyob rau hauv lub siab txo. Tom qab ntawd cov n-butanoic extract tau muab cais rau ntawm silica gel kem (200-300 mesh) siv cov gradient elution (CHCl3–MeOH, 10: 1 → 1: 1) kom muab yim feem. Fraction 6 (297 mg) tau siv rau kev cais HSCCC ntxiv thiab sib cais.
Kev xaiv ob-theem-Solvent Systems
Ob-theem hnyav tshuab raug xaiv raws li qhov sib faib coefficient (K) ntawm lub hom phiaj cov khoom hauv feem ntawm C. deserticola. Cov txiaj ntsig K tau txiav txim siab los ntawm LC kev tshuaj xyuas raws li hauv qab no: Cov nqi tsim nyog ntawm cov hmoov txhuv nplej siab (feem 6) tau yaj nyob rau theem qis ntawm cov kuab tshuaj thiab tshuaj xyuas los ntawm HPLC. Cov cheeb tsam ntawm lub ncov tau sau tseg raws li A1. Tom qab ntawd ib qho kev sib npaug ntawm cov theem sab sauv tau ntxiv rau qhov kev daws teeb meem thiab sib xyaw kom huv si kom ncav cuag qhov sib npaug sib npaug. Cov theem qis dua tau txheeb xyuas los ntawm HPLC dua. Cov cheeb tsam siab kawg tau sau tseg ua A2. K-tus nqi raug xam raws li qhov sib npaug hauv qab no: K=(A1 − A2)/A2 [15,16].
Kev Npaj Ob-Phase-Solvent System thiab Sample Solution
Nyob rau hauv txoj kev tshawb no, ob-theem hnyav system tsim los ntawm ethyl acetate-n-butanol- ethanol-water (40: 6: 6: 50, v / v / v / v) tau siv rau HSCCC sib cais. Txhua qhov hnyav tau ntxiv rau hauv qhov sib cais cais thiab sib npaug sib npaug ntawm chav tsev kub. Cov theem sab sauv thiab theem qis tau sib cais thiab degassed los ntawm sonication rau 30 min sai ua ntej siv. Cov qauv kev daws teeb meem tau npaj los ntawm kev sib cais 297 mg ntawm Fraction 6 hauv 15 ml ntawm theem qis ntawm ethyl acetate-n-butanol-ethanol-water (40: 6: 6: 50, v / v / v / v).
HSCCC Separation Procedure
HSCCC tau ua raws li hauv qab no. Lub multilayer coiled kem yog thawj zaug sau nrog cov organic nyob ruaj ruaj theem. Cov theem qis qis ntawm lub xov tooj ntawm tes tau muab tso rau hauv lub taub hau kawg ntawm kab ntawm qhov ntws ntawm 1.5 mL / min, thiab tib lub sijhawm, HSCCC apparatus tau khiav ntawm kev hloov pauv ceev ntawm 900 rpm. Tom qab hydrodynamic equilibrium tau tsim, raws li qhia los ntawm lub meej mobile theem eluting ntawm tus Tsov tus tw qhov hluav taws xob (kwv yees li ob teev tom qab), ib tug 15 mL qauv tov uas muaj 297 mg ntawm crude extract (feem 6) yog txhaj los ntawm lub txhaj valve. Cov effluent ntawm kem tau soj ntsuam tas li nyob rau hauv 254 nm. Plaub lub ncov feem ntau tau sau raws li chromatogram thiab tom qab ntawd evaporated nyob rau hauv qhov txo qis. Qhov kub ntawm cov cuab yeej tau teem rau ntawm 25 degree.
Kev txheeb xyuas HPLC thiab kev txheeb xyuas ntawm HSCCC Peak Fractions
Cov feem ntau ntawm HSCCC tau txheeb xyuas los ntawm HPLC-ELSD. Cov kem siv yog Accurasil C18 (250 mm × 4.6 mm, id 5 μm), theem mobile yog methanol-dej (30:70, v/v). Tus nqi ntws yog 1 mL / min. ELSD tau ua haujlwm raws li cov hauv qab no: Temp 45 degree, roj 1.6 L / min. Kev txheeb xyuas tus qauv ntawm plaub HSCCC qhov siab tshaj plaws yog ua los ntawm HR-ESI-MS, 1H, thiab 13C-NMR spectroscopy.
Kev Qhia Txog Tus Qauv
Tshooj I: HR-ESI-MS pom ntawm m/z 623.2001 (M−H)−, calcd rau C29H35O15, 623.1981. 1H-NMR (500 MHz, CD3OD) δ ppm: 1.09 (3H, d, J=6 Hz, CH3 ntawm rhamnose), 2.79 (2H, t, J=7.5 Hz, Ar-CH 2-), 4.37 (1H, d, J=8 Hz, H-1 ntawm qabzib), 5.18 (1H, d, J=1 Hz, H-1 } ntawm rhamnose), 6.27 (1H, d, J=15.5 Hz, Ar-CH=CH-), 7.59 (1H, d, J=15.5 Hz, Ar -CH=CH-), 6.5–7.1 (6H, aromatic H).13C-NMR (125 MHz, CD3OD) δ ppm: 131.5 (C-1), 117.2 (C{{65}) }), 146.2 (C-3), 144.7 (C-4), 116.4 (C-5), 121.3 (C-6), 72.4 (C- ), 36.6 (C- ), 127.7 (Caf-1), 115.3 (Caf-2), 146.9 (Caf-3), 149.8 (Caf-4), 116.6 (Caf{{{ 98}}), 123.2 (Caf-6), 168.3 (Caf- ), 114.8 (Caf- ), 148.1 (Caf- ), 104.3 (G-1), 76.1 (Glc{{116}) }), 81.7 (Glc-3), 70.5 (Glc-4), 76.3 (Glc-5), 62.4 (Glc-6), 103.1 (Rha{131} }), 72.3 (Rha{134}}), 72.1 (Rha-3), 73.9 (Rha{140}}), 70.7 (Rha{143}}), 18.5 (Rha{146}) }). Piv nrog rau cov ntaub ntawv muab hauv cov ntaub ntawv [17], feem Icorresponded rau acteoside.
Tshooj II: HR-ESI-MS pom ntawm m/z 623.1987 (M−H)−, calcd rau C29H35O15, 623.1981. 1H-NMR (500 MHz, CD3OD) δ ppm: 1.25 (3H, d, J=6.5 Hz, CH3 ntawm rhamnose), 2.78 (2H, t, J=7 Hz, Ar-CH2-), 4.33 (1H, d, J=8 Hz, H{29}} qabzib), 5.17 (1H, d, J {{ 33}} Hz, H-1 ntawm rhamnose), 6.28 (1H, d, J=15.5 Hz, Ar-CH=CH-), 7.56 (1H, d, J=15.5 Hz, Ar-CH=CH-), 6.5–7.0 (6H, aromatic H). 13C-NMR (125 MHz, CD3OD) δ ppm: 131.5 (C-1), 117.2 (C-2), 146.2 (C-3), 144.7 (C-4) ), 116.5 (C-5), 121.3 (C-6), 72.4 (C- ), 36.7 (C- ), 127.8 (Caf-1), 115.2 (Caf{{89}) } }), 146.8 (Caf-3), 149.7 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 169.2 (Caf- ), 115.0 (Caf- ), 147.3 (Caf- ), 104.5 (G-1), 75.5 (Glc-2), 84.2 (Glc-3), 70.1 (Glc{122}} ), 75.7 (Glc-5), 64.7 (Glc-6), 102.8 (Rha{131}}), 72.4 (Rha{134}}), 72.4 (Rha{137}} ), 74.1 (Rha{140}}), 70.5 (Rha{143}}), 17.9 (Rha{146}}). Cov ntaub ntawv 1H-NMR thiab 13C-NMR spectral tau pom zoo nrog cov isoacteoside raws li qhia hauv cov ntaub ntawv [18].
Tshooj III: HR-ESI-MS pom ntawm m/z 6{{108}}7.2032 (M−H)−, calcd rau C29H35O14, 607.2032. 1H-NMR (500 MHz, CD3OD) δ ppm: 1.09 (3H, d, J=6 Hz, CH3 ntawm rhamnose), 2.84 (2H, t, J=7.5 Hz, Ar-CH 2-), 4.37 (1H, d, J=8 Hz, H-1 ntawm qabzib), 5.19 (1H, d, J=1.5 Hz, H{{ 35}} ntawm rhamnose), 6.27 (1H, d, J=16 Hz, Ar-CH=CH-), 7.59 (1H, d, J=16 Hz, Ar-CH =CH-), 6.6–7.1 (7H, aromatic H). 13C-NMR (125 MHz, CD3OD) δ ppm: 130.8 (C-1), 116.2 (C-2), 130.9 (C-3), 156.8 (C-4) ), 130.8 (C-5), 116.2 (C-6), 72.4 (C- ), 36.4 (C- ), 127.8 (Caf-1), 114.8 (Caf{{88). } }), 149.8 (Caf-3), 146.9 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 168.3 (Caf- ), 115.4 (Caf- ), 148.0 (Caf- ), 104.3 (G-1), 76.3 (Glc-2), 81.7 (Glc-3), 70.4 (Glc-4 ), 76.1 (Glc-5), 62.5 (Glc-6), 103.0 (Rha{130}}), 72.3 (Rha{133}}), 72.2 (Rha{136}} ), 73.9 (Rha{139}}), 70.7 (Rha{142}}), 18.4 (Rha{145}}). Raws li cov ntaub ntawv [18], feem III sib raug rau syringalide A 3'- -L-rhamnopyranoside.
Tshooj IV: HR-ESI-MS pom ntawm m/z 665.21{{20}} (M−H)−, calcd rau C31H37O16, 665.2087. 1H-NMR (500 MHz, CD3OD) δ ppm: 1.09 (3H, d, J=6.5 Hz, CH3 ntawm rhamnose), 2.00 (3H, s, OAc), 2.72 (2H, t, J { {25}}.5 Hz, Ar-CH2-), 4.55 (1H, d, J=8 Hz, H-1 ntawm qabzib), 4.90 (1H, d, J { {37}} Hz, H-1 ntawm rhamnose), 6.29 (1H, d, J=15.5 Hz, Ar-CH=CH-), 7.62 (1H, d, J=15.5 Hz, Ar-CH=CH-), 6.5–7.0 (6H, aromatic H). 13C-NMR (125 MHz, CD3OD) δ ppm: 131.9 (C-1), 117.3 (C-2), 146.1 (C-3), 144.7 (C-4) ), 116.4 (C-5), 121.4 (C-6), 72.7 (C- ), 36.4 (C- ), 127.7 (Caf-1), 115.4 (Caf{{93}) } }), 146.9 (Caf-3), 149.9 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 168.1 (Caf- ), 114.7 (Caf- ), 148.2 (Caf- ), 101.8 (G-1), 75.3 (Glc-2), 80.3 (Glc-3), 70.8 (Glc{126}} ), 76.2 (Glc-5), 62.3 (Glc-6), 103.3 (Rha{135}}), 72.0 (Rha{138}}), 71.8 (Rha{141}} ), 73.7 (Rha{144}}), 70.8 (Rha-5), 18.5 (Rha{150}}), 171.5 (C=}O), 20.9 (OAC). Cov ntaub ntawv 1H-NMR thiab 13C-NMR spectral tau pom zoo nrog cov 2'-acetylacteoside [17].
Cov lus xaus
Ib txoj hauv kev HSCCC rau kev npaj sib cais thiab ua kom huv ntawm acteoside, isoacteoside, syringalide A 3'- -L-rhamnopyranoside thiab 2'-acetylacteoside los ntawmCistanches deserticola YC Matau tsim tsa. Txoj kev tshawb no qhia tau hais tias HSCCC yog cov txheej txheem muaj zog heev rau kev npaj sib cais thiab ua kom huv ntawm bioactive Cheebtsam los ntawm cov khoom cog. Tsis tas li ntawd, cov tebchaw tuaj yeem raug cais tawm ntawm qhov loj txaus nrog cov purities siab thiab tej zaum yuav raug siv los ua cov khoom siv rau chromatography lossis kev tshawb fawb bioactivity. Cov txheej txheem yog qhov ua tau, kev lag luam, thiab cov txheej txheem ua tau zoo rau kev npaj sai sai ntawm cov khoom siv ntuj tsim.
Kev lees paub
Txoj haujlwm no tau txais kev txhawb nqa los ntawm Program rau Changjiang Scholars thiab Innovative Research Team nyob rau hauv University (PCSIRT), Project of International Cooperation Plan los ntawm Ministry of Science thiab Technology ntawm Tuam Tshoj (2008DFB30070), thiab Scientific Program of Left Banner of Alashan ({{2 }}).
Cov ntaub ntawv
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