Novel Anti-Aging Benzoquinone Derivatives Los ntawm Onosma Bracteatum Phab Ntsa Ntu 2

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

2.3. Anti-Aging Activity nyob rau hauv K6001 Poov Strain

Kev tiv thaiv kev laus ntawm cov tshuaj benzoquinones cais (1-5) tau raug tshuaj xyuas los ntawm kev siv K6001 poov xab replicative lifespan bioassay system. Resveratrol (Res), uas paub zoo los tiv thaiv kev laus, tau siv los ua kev tswj xyuas qhov kev ntseeg siab ntawm bioassay system. Qhov zoo tshaj plaws concentration ntawm Res yog 10 μM nyob rau hauv cov poov xab relicative lifespan bioassay system raws li qhia nyob rau hauv peb cov ntawv tshaj tawm yav dhau los 【4-6】.cistanche tshuaj ntsuabRaws li pom nyob rau hauv daim duab 4, allomicrophyllone (1) ntawm 3 μM, ehretiquinone (2) thiab ehretiquinone C (4) ntawm 1 thiab 3 μM, thiab ehretiquinone D (5) ntawm 1 μM, nthuav tawm qhov tseem ceeb nce hauv qhov kev hloov pauv ntawm lub neej. K6001 hom poov xab, thaum ehretiquinone B (3) tsis ua haujlwm. Cov txiaj ntsig no tau qhia tias muaj peev xwm tiv thaiv kev laus ntawm benzoquinones (1, 2, 4, thiab 5) cais los ntawm O. bracteatum.

3. Kev sib tham

Hauv cov ntsiab lus ntawm kev laus thoob ntiaj teb, kev tshawb fawb thiab kev tsim cov tshuaj tiv thaiv kev laus los txhim kho lub neej zoo ntawm cov neeg laus thiab txo cov kab mob uas muaj hnub nyoog, yog qhov tseem ceeb heev.cistanche noov loj hlobTxoj kev tshawb no qhia txog kev sib cais, qauv elucidation, kev ua haujlwm lom neeg, thiab kev kawm luv SAR ntawm kev tiv thaiv kev laus benzoquinone derivatives los ntawm O.bracteatum1.

Thov nias ntawm no kom paub ntxiv

Hauv txoj kev tshawb no, ob qho paub (1 thiab 2) thiab peb qhov tshiab (3-5) benzoquinone derivatives raug cais tawm los ntawm ethyl acetate txheej qauv ntawm O.bracteatum raws li kev taw qhia ntawm K6001 poov xab replicaative lifespan bioassay. Cov qauv ntawm cov tshiab benzoquinones (3-5) tau elucidated los ntawm kev tsom xam spectroscopic. Cov paub benzoquinones, allomicrophyllone(1), thiab ehretiquinone(2) tau ntxuav thiab txheeb xyuas los ntawm kev sib piv cov ntaub ntawv spectroscopic nrog cov uas tau tshaj tawm hauv cov ntaub ntawv [17-19]. Yav dhau los, ob lub tebchaw tau tshaj tawm tias muaj kev tiv thaiv kev ua xua thiab tiv thaiv kev ua haujlwm, raws li [17,19]. Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, tsis muaj kev tshawb fawb txog kev tiv thaiv kev laus rau cov tebchaw no. Cov tshuaj sib cais no tau sib koom tib lub cev pob txha benzoquinone thiab lawv qhov sib txawv tau hloov kho ntawm C-11 thiab C-11 txoj haujlwm.

Cistanche tuaj yeem tiv thaiv kev laus

Kev ntsuam xyuas cov dej num lom neeg tau ua rau kev kawm txog cov qauv-kev sib raug zoo.cistanche salsa cov txiaj ntsigCov txiaj ntsig hauv daim duab 4 tau qhia tias cais allomicrophyllone (1), ehretiquinone(2), thiab ehretiquinones CD (4-5) ua rau lub neej ntev dua ntawm cov poov xab. Txawm li cas los xij, ehretiquinone B (3) uas muaj tib lub pob txha benzoquinone tab sis qhov kev hloov pauv txawv tsis ua haujlwm. Raws li kev tshawb fawb txog kev sib raug zoo-kev sib raug zoo (SAR) nyob rau hauv cov khoom sib cais no, peb tau xaus lus tias muaj methyl thiab exomethylene pawg ntawm C-9'yog qhov tseem ceeb rau kev tiv thaiv kev laus xws li ehretiquinone (2) thiab ehretiquinone C. (4). Kev hloov kho xws li hydroxylation ntawm C-11 / methyl pawg xws li hauv ehretiquinone B (3) ua rau poob haujlwm. Tsis tas li ntawd, kev sib piv ntawm ehretiquinone (2) thiab ehretiquinone C (4) nrog kev ua haujlwm zoo dua li cov allomicrophyllone (1) thiab ehretiquinone D (5) tau pom tias muaj cov pab pawg exomethylene ntawm C-9' ua ib qho tseem ceeb. lub luag haujlwm hauv kev tiv thaiv kev laus rau cov molecules. Ehretiquinone C(4) tau pom zoo los tiv thaiv kev laus dua li ntawm resveratrol vim hais tias qhov nruab nrab lifespan tus nqi ntawm 4 ntawm 1 uMis yog piv nrog resveratrol ntawm 10 uM uas qhia tib tus nqi.cistanche tubulosa noj redditYog li ntawd, ehretiquinone C(4) tuaj yeem suav hais tias yog ib qho kev cog lus ntawm cov hlau lead, thiab nws cov txheej txheem tiv thaiv kev laus yuav tsum tau kawm ntxiv. Tsis tas li ntawd, cov txiaj ntsig no tseem muab cov ntaub ntawv tseem ceeb rau kev tsim thiab tsim cov tshuaj tshiab los tiv thaiv kev laus. Txoj kev tshawb no yuav ua rau muaj txiaj ntsig rau kev tiv thaiv thiab kho cov kab mob muaj hnub nyoog. Cov txiaj ntsig kuj tau qhia tias O.bracteatum enriched nrog cov khoom xyaw tiv thaiv kev laus tuaj yeem siv los ua tshuaj lossis ua zaub mov ntxiv.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1.General

JASCO P{{0}} digital polarmeter tau siv los ntsuas qhov ntsuas qhov muag. Kev npaj siab ua kua chromatography (HPLC) nrog lub kaw lus nruab nrog ELITE P-230 twj thiab siv lub ntsuas UV. High-resolution huab hwm coj spectra (HR-ESI-TOF-MS) tau txais nrog Agilent Technologies 6224A qhov tseeb loj TOF LC / MS system. NMR spectra tau soj ntsuam nrog Bruker AVII-500 spectrometer thiab NMR tshuaj hloov pauv inδ (ppm) tau hais txog qhov hnyav ntawm bc 77.0 thiab 6H7.26 rau CDCl. Kem chromatography tau ua los ntawm kev siv silica gel ({14}} mesh, Yantai Chemical Industry Research Institute, Yantai, Tuam Tshoj) los yog thim rov qab theem C18 (Octadecylsilyl, ODS) silica gel (Cosmosil 75C18-OPN, Nacalai Tesque, Kyoto, Nyiv).

4.2. Cov khoom cog

Cov ntaub ntawv cog qoob loo tau yuav los ntawm Mansehra, Khyber Pakhtunkhwa, Pakistan, thiab pom tias yog Onosma bracteatum Phab ntsa los ntawm Dr. Zafar Ulah Zafar, Associate Professor, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. Daim ntawv pov thawj (20170220) ntawm cov nroj tsuag tau muab tso rau ntawm lub koom haum ntawm Materia Medica, Zhejiang University.

4.3. Extraction1 thiab cais tawm 1

Cov khoom siv cog qhuav (1.5 kg) yog hauv av rau hmoov thiab muab rho tawm nrog methanol (CH3OH) rau 3 hnub nrog rau kev sib txuas txuas ntxiv ntawm chav tsev kub. Cov tau txais crude extract (120} g) tau muab faib nruab nrab ntawm ethylacetate (EtOAc) thiab dej (H, O).Lub active ethylacetate txheej qauv (30 g) tau chromatographed ntawm ib tug silica gel qhib kab thiab eluted nrog n-hexane/CH, Cl(100:0,80:20, 50:50,0:100) thiab CH,Cl/CH3OH (98:2,95: 5, 90: 10 thiab 0: 100). Rau qhov seem tau txais. Fraction4 (8g) uas tau eluted nrog CH, Cl, / CH3OH (95: 5, 90: 10) tau ua tiav los ntawm ODSopen kem. CH3OH/H, O(40: 60, 50: 50, 55: 45, 60: 40, 70: 30, 90: 10 thiab 100: 0) tau siv los ua hnyav. Ntawm xya feem (Fr.{{46} }Fr.7) tau, Fr.2-Fr.4, tau muab cais ntxiv raws li hauv qab no:

Fr.4 (500 mg) raug cais los ntawm silica gel qhib kab thiab eluted nrog CH2Cl2/CH3OH (100:0, 99:1,98:2,97:3,95:5 ,90:10 thiab 0:100).Nib qauv (Fr.4-1-Fr.4-9) tau txais.Sample Fr.4-3 tau ua kom huv ntxiv los ntawm HPLC【C30- UG-5 (φ10 × 250 mm, Nomura Chemical), mobile theem∶ acetonitrile (CH3CN)/H2O(40:60), flow rate: 3 mL/min, and detector: 210nm] and yielded compound (1) (65 mg, tR =21.2 min).

Fr.3(113 mg) raug cais los ntawm kev siv ODS qhib kab thiab eluted nrog CH;OH/H2O(40:60, 45:55,50:50,55:45,60:40,80: 20 thiab 100:0).Kaum feem (Fr.3-1-Fr.3-10) tau txais, thiab Fr.{19}} tau ua kom huv ntxiv los ntawm HPLC【C30-UG{ {21}} (φ10 × 250 mm, Nomura Chemical), mobile theem∶methanol (CH3OH)/HO(68:32), flow rate: 3 mL/min, thiab detector: 210nm] and yielded compound (2) (20 mg, IR =20.3 min).

Fr.2(450 mg) tau raug silica gel qhib kab thiab eluted nrog CH2Cl2:CH3OH (100:0, 99:1,98: 2,97:3,95:5,90:1{{105}},0:100). cov feem (Fr.2-1-Fr.{{20}}) tau txais, thiab Fr.2-4(20 mg) tau raug ODS qhib kab chromatography nrog CH3OH/H2O(30}:70, 32:68,35:65,40:60 thiab 100∶O), thiab feem thib tsib ( 6.5 mg) tau purified los ntawm HPLC 【C30-UG-5} (φ10 × 250 mm, Nomura Chemical), theem mobile: acetonitrile(CH3CN)/H, O(35:65), flow rate: 3 mL/min, thiab ntes: 210 nm] rau cov khoom sib xyaw (3) (3.5 mg, t=31.0). Fr. 100:0) thiab qhov thib xya feem (11 mg) raug purified los ntawm HPLC 【C30-UG-5} (φ10 × 250 mm, Nomura Chemical), mobile theem∶acetonitrile (CH, CN)/HO (32:68), flow rate: 3 mL/min, thiab detector: 210 nm] to yield compound (4)(2.1 mg, tg=43.3).Fr.2-8(75 mg ) tau sib cais los ntawm ODS qhib kem siv CH3OH/H2O(20:80, 22:78.24:76.25:75.26:74,30:Z0.35:65.40:60 thiab 100:0) thiab ntu xya (10 mg) tau huv los ntawm HPLC 【C{107}}UG{108}} (φ10 × 250 mm, Nomura Chemical), mobile theem∶ acetonitrile (CH3CN)/H2O (30:70), flow rate: 3 mL/min, thiab detector : 210 nml rau vield compound (5)(4.3 mg, tn{120}}.3). Allomicrophyllone (1) . Cov hmoov daj; [ ]6 ntxiv rau 0.02 (c 0.57, CHCl3);High-resolution ESI-TOF-MS m/z 389.1359, xam rau C2H22O5Na (M plus Na) ntxiv rau 389.1359;'H NMR (500 MHz, CDCl3) {∶1bμ }}.83 (1H,dI=10.5 Hz,H-5^), 6.61(1H,d,J=8.6 Hz,H-6), 6.56(1H,d,J=2.8Hz,H-3),6.51(1H,dd,J=2.8, 8.6 Hz,H-5), 6.51 (1H,d, J=10.5 Hz, H-6'), 5.76(1H, d,J=15.9 Hz,H-8'), 5.67 (1H, d, J=15.9 Hz, H-7), 5.65(1H,d,J=7.1Hz,H-8), 3.77(1H, d,J=7.1Hz,H-7), 2.73(1H,d,J=19.2Hz,Hb-10), 2.50(1H,d,J{ {206}}.2 Hz,Ha-10), 1.69 (3H,s, H-11), 1.22(6H,s, H-10'& H-11 '); 13CNMR(125 MHz, CDCl3): 6c=195.8(C-1'), 193.1(C-4'), 150.0(C{230}}), 145.0(C-1), 143.3(C{{236}'), 139.4(C-5'), 138.6(C-6'), 131.8(C{245}) }), 127.7(C-2), 122.5(C-8), 122.0(C-7'), 117.6(C{257}}), 114.9(C{260) } }), 114.4(C-3), 80.7(C-3'), 71.0(C-9), 54.9 (C-2'), 39.1(C{ {275}}), 36.2(C-10), 29.8(C-10'), 29.8(C-11'), 22.6(C{287}}). re raug txheeb xyuas los ntawm kev sib piv ntawm MS, 'H NMR thiab 13C NMR spectra nrog rau kev sib hloov tshwj xeeb nrog cov ntaub ntawv [17,18]. Ehretiquinone (2) . Liab hmoov;[]16 ntxiv rau 1.01(c 0.12, CH3OH); high-resolution ESI-TOF-MS m/z 371.1254, xam rau C2.H20OANa (M plus Na) plus 371.1252;lH NMR (500MHz, CDCl3)∶δμ=6.83 (1H,d,J{{ 312}}.5Hz,H-5'),6.62(1H,d,J=8.6Hz,H-6),6.58(1H,d,J=2} .2 Hz,H-3), 6.53(1H,m,H-5), 6.52(1H,d,J=10.5 Hz,H-6') ,6.25(1H,d,J=16.0Hz,H-8),5.65(1H,d,J=6.2Hz,H-8), 5.50(1H ,d, J=16.0Hz,H-7), 5.04(1H,s,Hb-10), 4.97(1H,s,Ha-10'), 3.83 (1H,d, J=6.2Hz,H-7), 2.78(1H,d,I=19.1Hz,Hb-10), 2.52(1H,d ,J=19.1 Hz,Ha-10), 1.73(3H,s,H-11'), 1.70(3H,s,H-11);13CNMR ( 125MHz, CDCl): 6c=195.4(C-1), 193.2(C-4), 150.0(C-4), 145.1(C-1) 140.8 (C-9), 139.2(C-5), 138.8 (C-6'), 137.5(C-8'), 131.9(C{418) } }), 127.7(C-2), 124.1(C-7'), 122.7(C-8), 119.3(C-10'), 117.6(C{ {433}}), 114.9 (C-5), 114.4(C-3), 80.7(C-3'), 55.3(C-2'), 39.4( C-7), 36.3(C-10), 22.7(C-11), 18.3(C-11).Cov qauv raug txheeb xyuas los ntawm kev sib piv ntawm MS,'H NMR thiab 13C NMR spectra nrog rau kev sib hloov tshwj xeeb nrog cov ntaub ntawv [19].

Ehretiquinone B (3).Red hmoov; [ |6 plus 0.99(c 0.31, CH3OH); high-resolution ESI-TOF-MS m/z 387.1208, xam rau C22H20OsNa (M plus Na) ntxiv rau 387.1203; Cov ntaub ntawv rau 1H NMR thiab 13C NMR tau piav qhia hauv Cov Lus 1 thiab 2 raws li.

Ehretiquinone C (4). Liab hmoov; [ ] 16 plus 0.37 (c 0.12, CH, OH); high-resolution ESI-TOF-MS m/z 387.1174, xam rau C2H20O; Na (M plus Na) ntxiv rau 387.1174; Cov ntaub ntawv rau 1H NMR thiab 13C NMR tau piav qhia hauv Cov Lus 1 thiab 2 raws li.

Ehretiquinone D (5). Cov hmoov daj; [ l5 plus 0.51(c 0.41, CHCl3); high-resolution ESI-TOF-MS m/z 405.1300, xam rau C2, H, O, Na (M plus Na) ntxiv rau 405.1300; Cov ntaub ntawv rau HNMR thiab 13CNMR tau piav qhia hauv Cov Lus 1 thiab 2 raws li.

4.4. Lifespan AsSay/

Txoj kev bioassay siv rau txoj kev tshawb fawb los tiv thaiv kev laus tau piav qhia yav dhau los [4]. K6001 cov kab mob poov xab tau coj los ntawm YPGalactose lossis YPGlucose nruab nrab. YPGalactose nruab nrab tau npaj nrog 3 feem pua ​​​​galactose, 2 feem pua ​​​​hipolypeptone thiab 1 feem pua ​​​​cov poov xab extract thaum YPGlucose nruab nrab, muaj 2 feem pua ​​​​qabzib es tsis txhob galactose.cistanche แอมเวTxhawm rau npaj agar daim hlau, 2 feem pua ​​agar tau ntxiv rau qhov nruab nrab. Txhawm rau soj ntsuam cov qauv los ntawm bioassay, K6001 cov kab mob poov xab raug inoculated thiab incubated rau 24 teev nyob rau hauv cov poov xab peptone galactose nruab nrab nrog rau co. Tom qab 24 teev, qhov nruab nrab uas muaj cov kab mob poov xab yog centrifuged. Qhov tshwm sim ntawm cov poov xab pellets raug ntxuav nrog phosphate buffer tov (PBS) peb zaug thiab diluted. Tom qab dilution, cov hlwb raug xam nrog ib hemocytometer, thiab nyob ib ncig ntawm 4000 hlwb raug inoculated ntawm agar daim hlau uas muaj cov qauv uas muaj ntau yam concentrations. Tom qab ntawd, cov phiajcim tau muab tso rau hauv qhov cub ntawm 28 degree rau 2 hnub. Cov kab mob poov xab nyob rau hauv agar phaj tau pom nyob rau hauv lub tshuab kuaj kab mob tom qab 48 teev. Tag nrho ntawm 40 microcolonies los ntawm txhua lub phaj raug xaiv random, thiab tus naj npawb ntawm cov ntxhais hlwb raug suav thiab txheeb xyuas.

4.5. Kev txheeb cais

Qhov sib txawv tseem ceeb ntawm cov pab pawg tau txiav txim los ntawm ib txoj kev ANOVA nrog Dunnett ntau qhov kev sib piv. GraphPad Prism 5.0 software tau siv. Ap nqi<0.05 was="" considered="" statistically="">

5. Cov lus xaus

Peb qhov tshiab thiab ob lub npe hu ua benzoquinones raug cais tawm thiab tus cwj pwm los ntawm O. bracteatum raws li kev taw qhia ntawm K6001 poov xab replicaative lifespan assay. Cov qauv ntawm cov benzoquinones tshiab (3-5) tau piav qhia los ntawm kev txheeb xyuas spectroscopic, thiab cov neeg paub (1-2) tau txheeb xyuas los ntawm kev sib piv cov ntaub ntawv spectroscopic nrog cov hauv cov ntaub ntawv. Tag nrho cov benzoquinones cais tau raug soj ntsuam rau kev tiv thaiv kev laus los ntawm cov poov xab replicaative lifespan assay thiab 1,2,4 thiab 5 nthuav tawm cov haujlwm tseem ceeb los tiv thaiv kev laus.npaum li cas cistanche cojEhretiquinone C(4) tau pom tias muaj txiaj ntsig zoo tshaj plaws los tiv thaiv kev laus thiab yog li tuaj yeem suav tias yog cov khoom siv lead ua muaj peev xwm. Nws txoj kev tiv thaiv kev laus yuav tsum tau kawm ntxiv.

Cov khoom siv ntxiv:1D thiab 2D NMR spectra muaj nyob online ntawm http://www.mdpi.com/1420-3049/24/7/1428/s1.

Tus sau kev koom tes:UF tau ua qhov kev tshawb fawb tshuaj thiab sau cov ntawv sau. YP tau ua qhov kev ntsuam xyuas kev ua neej nyob. DD koom nrog hauv kev txheeb xyuas cov tebchaw. JQ tsim thiab saib xyuas qhov kev tshawb fawb no thiab kho cov ntawv sau.

Nyiaj txiag:Txoj haujlwm no tau txais kev txhawb nqa nyiaj txiag los ntawm National Key R&D Program ntawm Tuam Tshoj [No.2017YFE0117200] thiab NSFC [Grant Nos.21661140001,21877098, thiab 21572204].

Kev lees paub:Peb ua tsaug rau Michael Breitenbach ntawm Salzburg University, Austria rau qhov khoom plig ntawm S. cerevisiae poov xab K6001 strain. UF kuj lees paub txog kev txhawb nqa rau kev kawm kws kho mob los ntawm Lub Tsev Haujlwm Saib Xyuas Kev Kawm Qib Siab Pakistan.

Kev tsis sib haum xeeb ntawm kev txaus siab:Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.

Kab lus no tau muab rho tawm los ntawm Molecules 2019, 24, 1428; doi: 10.3390/molecules24071428 www.mdpi.com/journal/molecules