Cov Cim Zoo Ntawm Cistanches Herba Raws Li Antioxidant Spectrum-Effect Relationship

Abstract: Lub Hom Phiaj: Tsim ib txoj kev tshawb fawb rau cov cim zoo (Q-markers) ntawmCistanches Herba(CH) raws li qhovantioxidant spectrum-kev sib raug zoo, muab lub hauv paus rau kev kawm txog nws cov tshuaj pharmacological thiab kev tswj kom zoo. Txoj Kev: Ultra-high-performance kua chromatography (UPLC) tau siv los tsim cov ntiv tes ntiv tes rau 10 pawg ntawm CH. Lub cell oxidative kev puas tsuaj qauv tau tsim los siv H2O2, thiab lub peev xwm antioxidant ntawm CH tau txiav txim siab raws li ib nrab-qhov siab tshaj plaws inhibitory concentration ntawm reactive oxygen hom, nrog rau cov qib ntawmsuperoxidedismutasethiabcatalaseraws lipharmacological ntsuas. Kev sib raug zoo ntawm spectrum-zoo tau tsim los ntawm kev tsom xam cov ntsiab lus tseem ceeb, Pearson correlation tsom xam, thiab ib feem tsawg squares regression tsom xam. Covactive Cheebtsamraug tsim txompharmacological validation. 

Cov txiaj ntsig:Cov ntawv ntiv tes rau CH tau tsim los siv UPLC, txheeb xyuas 17 qhov siab tshaj plaws. Raws li spectrum-effect correlation tsom xam, cuaj Cheebtsam raug xaiv. Cov txiaj ntsig pharmacological tau qhia tias geniposidic acid, 8-epiloganic acid,echinacoside cov tshuaj,acteoside, isoacteoside, thiabtubuloside Anthuav tawm cov kev ua antioxidant hauv qhov concentration-dependent yam.

Cov ntsiab lus: Txoj kev tshawb no tshawb nrhiav Q-cov cim rau CH raws li kev sib raug zoo ntawm kev sib raug zoo, muab cov ntaub ntawv siv rau nws cov tshuaj siv tshuaj hauv paus thiab kev txhim kho tus qauv zoo.

Ntsiab lus:Cistanches Herba; Q-kos npe;kev sib raug zoo spectrum; antioxidant

Cistanche deserticola yog lub qia qhuav nrog scaly nplooj ntawm Cistanche deserticola YCMa los yog Cistanche tubulosa (Schenk) Wight, ib tsob nroj ntawm Cistanche deserticola tsev neeg orbaceae. Nws yog sov nyob rau hauv qhov, qab zib thiab ntsev nyob rau hauv saj; nws rov qab mus rau lub raum thiab txoj hnyuv loj meridians; nws muaj cov teebmeem ntawm nourishing raum yang, replenishing essence thiab ntshav, moistening cov hnyuv thiab laxative, thiab yog ib qho tseem ceeb tshuaj rau tonifying lub raum thiab replenishing essence [2-3]. Lub raum khaws cov essence, thiab cov ntsiab lus thiab cov ntshav los ntawm tib qhov chaw. Yog tias cov ntsiab lus thiab cov ntshav tsis txaus, nws yuav cuam tshuam rau tag nrho lub neej kev ua ub no ntawm tib neeg lub cev; cov pob txha tsim cov essence yog khaws cia rau hauv cov pob txha, thiab lub hlwb yog cov pob txha hauv hiav txwv. Yog tias lub raum tsis txaus, cov pob txha yuav poob lawv cov khoom noj thiab lub hlwb yuav tsis txaus, ua rau cov pob txha degenerative hloov xws li xoob kev ntxhib los mos, hniav poob, nco tsis tau, dementia, thiab tsis muaj zog tiv thaiv kab mob [4-6]. Cistanche deserticola muaj phenylethanoid glycosides, iridoids, lignans thiab lwm yam khoom siv, xws li echinacoside, verbascoside, verbascoside, genipinic acid, thiab 8-epistryanic acid. , tubulin A, etc. [7]. Kev tswj xyuas zoo tam sim no ntawm Cistanche deserticola xyoo 2020 tsab ntawm "Pharmacopoeia ntawm Cov Neeg Sawv Cev ntawm Tuam Tshoj" tsuas yog teev cov ntsiab lus ntawm echinacoside thiab verbascoside, uas tsis tuaj yeem cuam tshuam tag nrho cov ntsiab lus thiab ntau lub hom phiaj ntawm cov tshuaj suav tshuaj. , uas tsis tsim nyog rau nws cov kev tswj xyuas zoo thiab kev ntsuam xyuas.

Txhawm rau txhim kho qib kev tswj hwm zoo ntawm cov tshuaj suav tshuaj thiab ua kom zoo tshaj qhov kev ntsuas zoo ntawm cov tshuaj suav tshuaj, Academician Liu Changxiao tau npaj lub tswv yim tshiab ntawm cov cim zoo (Q-marker) ntawm cov tshuaj suav tshuaj [8]. Ntawm lawv, "kev ua tau zoo" yog lub hauv paus tseem ceeb hauv "Tsib Txoj Cai" ntawm Q-kos npe [9] thiab tseem yog lub hauv paus ntawm kev ntsuam xyuas zoo ntawm cov tshuaj suav tshuaj.

Raws li kev tshawb fawb tam sim no ntawm kev tsis sib raug zoo ntawm cov qauv kev ua tau zoo thiab kev siv tshuaj, kev tshawb nrhiav Q-cov cim ntawm cov tshuaj suav tshuaj thiab tsim kom muaj cov qauv kev tshawb fawb thiab tsim nyog zoo dua yog qhov tseem ceeb rau kev txhim kho cov tshuaj suav tshuaj zoo. Txoj kev sib raug zoo ntawm kev sib raug zoo txuas cov ntiv tes chromatographic peaks ntawm cov tshuaj suav tshuaj suav nrog cov ntaub ntawv pharmacodynamic kom pom cov tshuaj zoo ntawm cov tshuaj suav tshuaj, yog li qhia txog qhov zoo ntawm cov tshuaj suav tshuaj [10]. Kev tsim cov qauv kev ua lej spectral tuaj yeem qhia meej txog QMarker ntawm cov tshuaj suav tshuaj los ntawm "kev pom zoo" kev xav, muab kev siv ntau ntxiv rau kev tswj xyuas cov tshuaj suav tshuaj zoo tom qab, tsim cov qauv zoo, thiab tshuaj xyuas cov khoom xyaw nquag. Txoj kev tshawb no siv cov ntiv tes ua ke nrog ib nrab-inhibition tus nqi ntawm cov pa oxygen reactive (ROS IC50), cov ntsiab lus ntawm superoxide dismutase (SOD) thiab catalase (CAT) los kawm txog kev sib raug zoo ntawm kev sib raug zoo ntawm cov tshuaj suav tshuaj, thiab siv cov tshuaj ntsuam xyuas tseem ceeb. (PCA)), Pearson correlation analysis (PCC) thiab ib feem tsawg squares regression tsom xam (PLS) kom nkag siab txog kev txheeb xyuas Q-kos npe ntawm Cistanche deserticola los muab lub hauv paus rau nws cov kev tswj kom zoo.

1 khoom

1.1 Qauv

Cistanche deserticola tau sau los ntawm Yutian County, Xinjiang, thiab Cistanche deserticola Good Agricultural Practice (GAP) cog hauv Inner Mongolia. Nws tau txheeb xyuas los ntawm tus kws tshawb fawb Zhang Tiejun ntawm Tianjin Pharmaceutical Research Institute Co., Ltd. li Cistanche tubulosa (Schenk) Wight thiab Cistanche deserticola.

YC Ma, cov ncauj lus kom ntxaws qhia hauv Table 1.

1.2 Test tshuaj

Echinaceaside (batch tooj: 111670-201907, purity: 91.8%) thiab verbascoside (batch tooj: 111530-201914, purity: 95.2%) yog ob qho tib si yuav los ntawm Tuam Tshoj lub koom haum ntawm Food and Drug Control; isorescoside (batch tooj: 61303-13 -7), salidroside (batch tooj: 10338-51-9), 8-epistrychnoic acid (batch tooj: 82509-41-9), genipinic acid (batch tooj: Z24A10X95926) , tuberose B (Batch tooj: 112516-04-08), 2-acetyl eugenol glycoside (Batch tooj: 94492-24-7) tau muas los ntawm Shanghai Yuanye Biotechnology Co., Ltd., purity Ntau dua los yog sib npaug rau 98% yog '; Cistanche glycoside A (Batch tooj: YUAV TSUM -20062806, purity: 98.51%), floridin A (batch naj npawb: MUST19110102, purity: 98.57%) tau muas los ntawm Chengdu Mansite Biotechnology Co., Ltd.; chromatographically ntshiab acetonitrile thiab formic acid tau muas los ntawm Tianjin Concord Technology Co., Ltd. Lub tuam txhab; Purified Dej (Hangzhou Wahaha Group Co., Ltd.). Vitamin E (VE, V8010, Beijing Solarbio Company); dimethyl sulfoxide (DMSO, D2660-100ML, Sigma Company ntawm Tebchaws Meskas); CellTiter 96® AQueous ib qho kev daws teeb meem cell proliferation detection kit (G3580, Promega Company of the United States); CAT kuaj pom Cov khoom siv (S0051), SOD cov khoom siv tshawb nrhiav kev ua haujlwm (S0101M), thiab cov khoom siv kuaj pom ROS (S0033S) tau yuav los ntawm Shanghai Biyuntian Biotechnology Co., Ltd.

1.3 Cov cuab yeej

Waters H-class-PDA ultra-siab ua kua chromatograph (Waters Company, USA); Sartorius BT25S hluav taws xob tshuav nyiaj li cas (Sartorius Company, Lub teb chaws Yelemees); H1650 high-speed desktop centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); SpectraMax M5

Microplate nyeem ntawv (Meigu Molecular Instrument Co., Ltd.); WYS-41XDY biological microscope (Tianjin Micro-Instrument Optical Instrument Co., Ltd.).

1.4 cev

Tib neeg lub raum hlwb HEK 293 hlwb tau siv rau hauv qhov kev sim no thiab tau yuav los ntawm lub txhab nyiaj cell ntawm Tuam Tshoj Academy of Sciences.

2 txoj kev

2.1 Tsim daim ntawv qhia ntiv tes

2.1.1 Kev npaj cov tshuaj ntsuam xyuas: hnyav 2 g ntawm Cistanche deserticola cov khoom siv tshuaj, ntxiv 10 zaug ntawm cov dej, cua sov thiab reflux 3 zaug, 1 h txhua zaus, centrifuge ntawm 1.0 × 105r·min-1 rau 15 min (centrifugal radius yog 300 mm), Sib xyaw cov supernatants thiab mloog zoo kom ua cov tshuaj hauv Tshuag nrog cov concentration ntawm 100 mg·mL-1, dilute nws 10 zaug, thiab lim nws los ntawm 0.22 µm microporous lim membrane kom tau txais nws.

2.1.2 Kev npaj cov tshuaj sib xyaw ua ke

Noj 8-epistrrychnic acid, salidroside, echinaceaside, cistanche glycoside A, genipinic acid, verbascoside, anthocyanoside A, isorbascoside, thiab 2'-acetyl eugenol. Glycoside thiab angioside B cov tshuaj siv tau raug ntsuas hauv qhov tsim nyog thiab ntsuas qhov tseeb. Ntxiv 50% cov tshuaj methanol rau yaj thiab npaj cov tshuaj sib xyaw ua ke nrog cov concentration ntawm 20 µg·mL-1.

2.1.3 Chromatographic tej yam kev mob

ACQUITY UPLC®BEH C18 kem (100 mm × 2.1 mm, 1.7 µm); mobile theem yog acetonitrile (A) ~ 0.1% formic acid tov (B), gradient elution program (0 ~ 6 min, 5% ~ 8% A; 6 ~ 10 min, 8% ~ 15% A 10 ~ 13 min, 15% ~ 19% A, 13 ~ 20 min, 19% ~ 25% A); nrhiav wavelength yog 237 nm; tus nqi ntws yog 0.4 mL·min -1; sab kub yog 40 degree; Kev txhaj tshuaj yog 3 µL.

2.1.4 Reference ncov xaiv

Nyob rau hauv ultra-performance kua chromatography (UPLC) chromatogram ntawm Cistanche deserticola, lub ncov nrog lub ncov loj tshaj plaws cheeb tsam piv, nruab nrab lub sij hawm tuav, thiab kev sib cais zoo tau xaiv raws li lub ncov ntawm cov ntiv tes spectrum. 2.1.5 Methodological kev soj ntsuam Precision test: Coj tus qauv T1 thiab npaj cov kev xeem daws raws li txoj kev hauv qab 2.1.1. Txhaj tus qauv 6 zaug tsis tu ncua thiab sau tus ntiv tes. Cov txiaj ntsig tau pom tias qhov txheeb ze thaj tsam RSD ntawm txhua lub ncov yog 0.10% ~ 4.61%. , RSD ntawm cov txheeb ze tuav lub sij hawm yog 0.08% ~ 1.32%, qhia tau hais tias cov cuab yeej muaj precision zoo.

Kev xeem rov ua dua: Siv cov qauv T1 thiab npaj 6 cov qauv ntawm cov kev sim tshuaj sib xyaw ua ke raws li cov qauv hauv qab 2.1.1, thiab txhaj cov qauv txuas ntxiv mus. Cov txiaj ntsig tau pom tias qhov txheeb ze thaj tsam RSD ntawm txhua qhov chromatographic ncov yog 1.35% ~ 4.80%, thiab lub sij hawm tuav tus txheeb ze RSD yog 0.10% ~ 1.80%, uas ua tau raws li qhov yuav tsum tau muaj ntawm tus ntiv tes thiab qhia tau tias qhov rov ua dua. ntawm qhov ntsuas tau zoo.

Kev ntsuas kev ruaj ntseg: Siv cov qauv T1 thiab npaj cov kev ntsuas ntsuas raws li txoj hauv kev hauv qab 2.1.1, txhaj cov qauv rau kev sim ntawm 0, 2, 4, 8, thiab 12 teev, thiab sau tus ntiv tes. Qhov xam RSD ntawm thaj chaw txheeb ze ntawm txhua qhov chromatographic ncov yog 0.53% ~ 4.82%, thiab RSD ntawm tus txheeb ze tuav lub sij hawm yog 0.09% ~ 2.17%, uas ua tau raws li qhov yuav tsum tau ua ntawm tus ntiv tes thiab qhia tias cov khoom kuaj tau ruaj khov hauv 12 teev.

2.2 Kev tshawb fawb txog cov nyhuv antioxidant ntawm Cistanche deserticola

2.2.1 Kev npaj ntawm Cistanche deserticola niam cawv:Coj niam cawv nyob rau hauv nqe lus 2.1.1, lim thiab kom tsis muaj menyuam, thiab dilute nws nrog kab lis kev cai nruab nrab mus rau qhov xav tau concentration ua ntej siv.

2.2.2 Cov teebmeem ntawm Cistanche deserticola thiab kev tswj zoo VE ntawm kev muaj peev xwm ntawm HEK 293 hlwb

Teeb tsa cov pab pawg ib txwm (cell ncua kev kawm), Cistanche deserticola pawg tswj hwm (25, 50, 100, 200, 400 µg·mL–1), VE pawg (20, 40, 60, 80, 100 µmol·L–1), Txhua Teeb 6 qhov sib npaug ntawm txhua qhov kev xav thiab kab lis kev cai rau 24 teev. Ntxiv 20 µL ntawm CellTiter 96® AQueous ib qho tshuaj reagent rau txhua qhov dej txhawm rau txheeb xyuas cov cell muaj peev xwm. Txuas ntxiv culturing rau 2 teev. Siv tus nyeem ntawv microplate txhawm rau txheeb xyuas tus nqi nqus (OD) ntawm txhua qhov dej ntawm 490 nm. Xam cov cell ciaj sia taus raws li cov qauv (1). Ciaj sia taus tus nqi (%)=(OD sim pawg/OD dawb paug pab pawg) × 100% (1)

2.2.3 Cov nyhuv ntawm Cistanche deserticola ntawm ROS ntxias los ntawm H2O2 hauv HEK 293 hlwb

Cov hlwb tau muab faib ua pawg (cell suspension) thiab Cistanche deserticola pawg thawj coj (6.25, 12.5, 25, 50, 100 µg·mL–1 Cistanche deserticola thiab 60 µm·L–1 H2O2), thiab coj mus rau 24 teev. Ua raws li cov kauj ruam ntawm cov khoom siv tshawb nrhiav ROS rau kev ua haujlwm tom ntej, ncaj qha saib cov xov tooj ntawm tes los ntawm lub tshuab tsom, thiab siv tus nyeem ntawv microplate txhawm rau txheeb xyuas qhov kev siv fluorescence ntawm txhua pab pawg. Parameter teeb tsa siv qhov kev ua kom muaj zog ntawm 488 nm thiab emission wavelength ntawm 525 nm. Qhov kev sim tau rov ua dua peb zaug.

2.2.4 Cov nyhuv ntawm Cistanche deserticola ntawm H2O2-induced SOD ntawm HEK 293 hlwb

Faib cov hlwb rau hauv ib pab pawg (cell suspension) thiab pawg qauv

(60 µm·L–1 H2O2), VE pawg (60 µm·L–1 VE thiab 60 µm·L–1 H2O2), Cistanche deserticola pawg tswj hwm (100 µg·mL–1 Cistanche deserticola thiab 60 µm·L–1 H2O2 ), sau cov hlwb tom qab 24 teev ntawm kab lis kev cai, ntxiv SOD cov qauv npaj tov rau lyse cov hlwb, centrifuge ntawm 4 degree, 12000 r·min-1 rau 5 min (centrifugal radius yog 300 mm), coj tus supernatant raws li cov qauv uas yuav tsum tau sim, ua raws li cov khoom siv SOD nrhiav tau raws li cov kauj ruam hauv qab no. Qhov kev sim tau rov ua dua peb zaug.

2.2.5 Cov nyhuv ntawm Cistanche deserticola ntawm H2O2-induced CAT hauv HEK 293 hlwb

Ua raws li cov qauv hauv qab 2.2.4 thiab kev tswj hwm pab pawg, ntxiv cov tshuaj lysis, centrifuge ntawm 4 degree , 12000 r·min -1 rau 10 feeb (centrifuge radius yog 300 mm), coj tus supernatant, thiab ua raws li cov kauj ruam ntawm CAT kuaj cov khoom siv. Kev ua haujlwm tom qab. Qhov kev sim tau rov ua dua peb zaug.

2.2.6 Kev txheeb xyuas

SPSS 26.0 software tau siv rau kev txheeb cais. Txhua pawg ntawm cov ntaub ntawv tau sawv cev los ntawm (± ± (). Ib txoj kev tsom xam ntawm qhov sib txawv tau ua rau ntawm cov ntaub ntawv uas ua raws li qhov kev faib tawm ib txwm muaj. tsis sib npaug, Dunnett's T3 txoj kev siv GraphPad Prism 8 tau siv rau kev teeb tsa, thiab P<0.05 was considered as a statistically significant difference.

2.3 Spectrum-effect correlation tsom xam ntawm Cistanche deserticola antioxidant

2.3.1 PCA Cov cheeb tsam siab tshaj plaws ntawm qhov siab tshaj plaws ntawm 10 batches ntawm Cistanche deserticola uas tau tsim cov ntiv tes tau tsim los ntawm Z tus nqi, thiab ua rau hauv 10 × 17 cov ntaub ntawv txiav txim matrix, uas tau raug xa mus rau hauv SPSS 26.0 software rau PCA.

2.3.2 PCC Siv SPSS 26.

Kev ntsuam xyuas qhov tseem ceeb siv qhov kev sim ob sab (T) nrog P<0.05 as the standard for statistically significant differences.

2.3.3 PLS Ntshuam cov kev sim ntawm Cistanche deserticola tus qauv peak cheeb tsam thiab antioxidant rau hauv SIMCA-P 14.1 software, teem lub ncov cheeb tsam raws li qhov sib txawv X, thiab teem lub pharmacodynamic Performance index raws li Y sib txawv los ua PLS.

2.4 Cistanche deserticola Q-marker antioxidant kev ua pov thawj

2.4.1 Kev npaj ntawm cov khoom xyaw active niam cawv

Ua kom hnyav qhov tsim nyog ntawm genipinic acid, 8-epistrichnic acid, echinaceaside, verbascoside, isorebascoside, thiab anthocyanoside A, thiab ntxiv DMSO kom yaj rau hauv 10 mmol·L-1 rau kev tswj. cov khoom tov.

2.4.2 Cov nyhuv ntawm cov khoom xyaw nquag ntawm HEK 293 cytotoxicity

Tom qab HEK 293 hlwb raug incubated rau 24 teev, 100 µL ntawm active ingredient tov nyob rau hauv cov khoom 2.4.1 (0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 μL) ntawm mol·L sib txawv concentrations tau ntxiv rau txhua qhov dej, thiab stimulation tau txuas ntxiv mus. CellTiter 96® AQueous ib qho tshuaj reagent tau ntxiv rau hauv lub hlwb 24 teev tom qab txhawm rau txhawm rau txheeb xyuas qhov muaj peev xwm ntawm tes.

2.4.3 Cov nyhuv antioxidant ntawm cov khoom xyaw nquag ntawm H2O2-induced HEK 293 hlwb

Cells tau muab faib ua pawg raws li hauv qab no: pawg ib txwm, pawg qauv, VE pawg tau muab faib ua pawg hauv 2.2.4, pawg sim (0.781, 1.563, 3.125 µm·L–1 cov khoom xyaw nquag thiab 60 µm·L–1 H2O2), kab lis kev cai rau 24 teev. Txiav txim siab intracellular ROS qib, SOD thiab CAT enzyme cov ntsiab lus raws li txoj hauv kev hauv qab 2.2.3, 2.2.4 thiab 2.2.5 raws.

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