Iridoid Thiab Acyclic Monoterpene Glycosides, Kankanosides L, M, N, O, Thiab P Los ntawm Cistanche Tubulosa

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Cistanche tubulosa(SCHRENK) R. WIGHT (Orobanchaceae) yog ib tsob nroj uas muaj kab mob muaj hnub nyoog loj hlob ntawm cov hauv paus hniav ntawm Salvadora lossis Calotropis hom, faib nyob rau hauv North Africa, Arabia, thiab Asian lub teb chaws. siv rau kev kho mob ntawm impotence, sterility, lumbago, thiab lub cev tsis muaj zog nrog rau kev txhawb nqa ntawm cov ntshav ncig.1,2) Thaum lub sijhawm peb kawm txog bioactive constituents los ntawm stems ntawm Cistanche tubulosa, 3-6) peb yav tas los qhia nees nkaum plaub phenylethanoid aminoglycosides suav nrog kankanosides H1, H2, I, J1, J2, K1, thiab K2, thiab ob lub acylated oligosugars los ntawm cov stems tshiab ntawm Cistanchetubulosa.5,6) Tsis tas li ntawd, tus thawj xib fwbphenylethanoid glycosides,echinacoside, acteoside,thiab isoacteoside, tau pom tias inhibit qhov nce hauv serum aspartate aminotransferase (sAST) thiab alanine aminotransferase (sALT) qib hauv cov nas lub siab raug mob vim D-galactosamine (D-GalN) / ​​lipopolysaccharide ntawm koob tshuaj 25-100 mg / kg ib os ( po).Structural requirements of the phenylethanoid glycosides for the hepatoprotective act also elucidated.5) Hauv qhov kev tshawb fawb txuas ntxiv no ntawm cov khoom siv hauv cov stems tshiab ntawm Cistanchetubulosa, peb ntxiv cais kaum ib iridoid glycosides suav nrog kankanosides L (2), thiab N (3), xya acyclic monoterpene glycosides suav nrog kankanosides O (4) thiab P (5), peb phenylpropanoids, thiab plaub lignans. Daim ntawv no hais txog kev sib cais thiab qauv elucidation ntawm tsib lub tebchaw tshiab (1-5).

Cov stems tshiab ntawm C. tubulosa (cog nyob rau hauv Urumuqi, Xin jiang Province, Tuam Tshoj) tau muab rho tawm nrog methanol nyob rau hauv reflux kom tau ib tug methanolic extract (8.36 feem pua ​​ntawm cov tshiab stems). Los ntawm cov methanolic extract, H2O- thiab MeOH-eluted feem (5.63 feem pua ​​thiab 2.73 feem pua) tau txais los ntawm Diaion HP-20 kab chromatography (H2O→MeOH) raws li tau piav yav tas los.5) MeOH-eluted feem yog sub jected rau SiO2 thiab ODS kab chromatographies thiab kawg HPLC los muab kankanosides L (1, 0.0026 feem pua ​​, M (2, 0.{{ 27}001% ), N (3, 0). , O (4, 0). }. 0. (10, 0.0056%), 8- epiloganic acid3,8) (11, 0.0023%), 8-epideoxyloganic acid3,7) (12, 0.0004%) , geniposidic acid3,7) (13, 0.0040 feem pua ​​), kankanoside E3) (14, 0.0026 feem pua ​​), (2E,6Z)-8-b D-glucopyranosyloxy-2,6-di methyl-2,6-octadienoic acid3,9) (15, 31.0 mg, 0.0014%), (2E,6E){{95 }}, 7-dimethyl-8-hydroxyoctadien-1-yl-ObD-glu copyranoside10) (16, 0.0082%), 8-hydroxygeraniol 8-Ob- D-glucopyranoside11 ) (17, 0.0044%), betualbuside A12) (18, 0.0004 feem pua), coniferin13) (19, 0.0002 feem pua), syringin13) (20, 0.0015%), sinapic aldehyde {{128-128}} 0.0001 feem pua ​​, ( )-pinoresinol ObD-glucopyranoside4,8,15) (22, 0.0010 feem pua), eucommin A16) (23, 0.0002 feem pua), isoeucommin A17) (24, 0.0010 feem pua-D), thiab () glucopyra noside4,8,18) (25, 0.0044 feem pua).

Cistanche tubulosatxhim kho kev ua haujlwm ntawm kev sib deev

Cov qauv ntawm Kankanosides L (1), M (2), thiab N (3)

Kankanoside L (1) tau txais raws li cov hmoov dawb nrog qhov tsis zoo kho qhov muag ([a]D 26 45.7 hauv MeOH). Nws IR spectrum tau pom muaj zog nqus ntawm 3433 thiab 1{{20}}80 cm 1 qhia txog glycoside moiety. Lub nrawm atom bombardment (FAB)-MS ntawm 1 khiav hauv qhov zoo- thiab tsis zoo-ion hom pom quasimolecular ion peaks ntawm m / z 371 [M Na] thiab 347 [MH], raws li, thiab cov qauv molecular tau txiav txim siab li C15H24O9. los ntawm high-resolution FAB-MS ntsuas. Acid hydrolysis ntawm 1 nrog 1.0 M hydrochloric acid (HCl) liberated D-glucose, uas tau txheeb xyuas los ntawm HPLC kev tshuaj xyuas siv lub ntsuas qhov muag rotation.3-6) Lub 1 H- thiab 13C-NMR spectra ntawm 1 (CD3OD, Tables 1, 2), uas tau muab los ntawm ntau yam NMR thwmsim, 19) pom cov cim taw qhia rau plaub methylene [d 1.43 (1H, br dd, J ca. 5, 13 Hz, 4a-H), 1.73 (1H, br dd, J. ca. 12, 14 Hz, 6a-H), 1.85 (1H, m, 4b-H), 1.93 (1H, br dd, J= ca. 8, 14 Hz, 6b-H), 3.50 ( 1H, ddd, J 2.4, 12.5, 13.0 Hz, 3a-H), 3.83 (1H, br dd, J=ca. 5, 13 Hz, 3b-H), 3.78, 4.12 (1H txhua, ob qho tib si d, J 13.1 Hz, 10-H2)], ob methines [d 2.15 (1H, dd, J 7.2, 8.9 Hz, 9-H) thiab 2.23 (1H, m, 5-} H)], thiab ib pawg acetal [d 4.81 (1H, d, J 8.9 Hz, 1-H)] ua ke nrog b-glucopyranosyl moiety [d 4.70 (d, J 7.9 Hz, 1 -) H)]. Raws li pom nyob rau hauv daim duab 1, 1 H–1H correlation spectroscopy (1 H–1 H COSY) kev sim ntawm 1 qhia tias muaj ib feem ntawm cov qauv sau nyob rau hauv bold kab. Hauv kev sim heteronuclear multiple-bond correlation (HMBC) ntawm 1, kev sib raug ntev ntev tau pom ntawm cov protons thiab carbons hauv qab no (1-H thiab 3-C, 8-C; {{ 124}}H thiab 1-C; 7-H thiab 8-C, 10-C; 9-H thiab 8-C; {{131} }}H2 thiab 7-C, 8-C; 1 -H thiab 1-C) raws li qhia nyob rau hauv daim duab 1. Tom ntej no, tus txheeb ze stereostructure ntawm 1 yog yus muaj los ntawm theem-sensitive nuclear Overhauser enhancement spectroscopy (phase-sensitive NOESY) kev sim, uas qhia NOE kev sib raug zoo ntawm cov proton khub hauv qab no (1-H thiab 3a H; 3a-H thiab 4a-H; 3b-H thiab 4b-H; 4b-H thiab 5-H, 9-H; 5- H thiab 6b-H, 9-H; 6a-H thiab 7-H; 7-H thiab 10-H2), raws li qhia nyob rau hauv daim duab 1. Lub 1 H- thiab 13C-NMR spectra ntawm 1 yog superimposable rau cov thawj coj iridoid constituent 6-deoxycatalpol (6), tshwj tsis yog rau cov teeb liab vim mus rau saturated d-lactol moiety. Thaum kawg, hydrogenation ntawm 6 muab 1, kom lub stereostructure ntawm kankanoside L tau elucidated los ua 3, 4- dihydro-6-deoxycatalpol (1).

Kankanoside M (2) tau txais raws li cov hmoov dawb nrog qhov tsis zoo ntawm kev sib hloov kho qhov muag ([a]D 26 18.7 hauv MeOH). IR spectrum ntawm 2 tau pom cov khoom nqus ntawm 3433, 1736, 1655, thiab 1076 cm 1 ascribable rau hydroxyl, d-lactone, olefin, thiab ether moieties. Qhov zoo-ion FAB-MS spectrum ntawm 2 tau pom quasimolecular ion ncov ntawm m / z 353 [M Na] , thiab cov mis mos molecular tau txiav txim siab li C15H22O8 los ntawm high-resolution positive-ion FAB-MS ntsuas. Acid hydrolysis ntawm 2 nrog 1.0 M HCl liberated D-glucose. Lub 1 H- thiab 13C NMR spectra ntawm 2 (CD3OD, Tables 1, 2) pom cov cim qhia tau rau plaub methylene [d 1.66, 2.11 (1H txhua, ob qho tib si m, 4-H2), 2.15, 2.75 (1H txhua. , ob m, 6-H2), 4.29 (1H, ddd, J 2.8, 8.4, 14.3 Hz, 3b-H), 4.32, 4.51 (1H txhua, ob qho tib si d, J 13.1 Hz, 10- H2), 4.35 (1H, ddd, J 3.1, 6.7, 14.3 Hz, 3a-H)], ob methines [d 2.97 (1H, m, 5-H), 3.82 (1H, br s, {{ 84}}H)], ib qho olefin [d 5.94 (1H, m, 7-H)], thiab ib pawg lactone saturated (d C 174.9) ua ke nrog bD-glucopyranosyl moiety [d 4.32 (1H, d , J 7.9 Hz, 1 -H)]. Raws li pom hauv daim duab 1, qhov kev sim 1 H–1H COZY ntawm 2 tau qhia tias muaj ib feem ntawm cov qauv sau nyob rau hauv cov kab bold thiab, hauv HMBC kev sim, kev sib raug zoo ntev tau pom ntawm cov proton thiab carbon khub hauv qab no ({105 }}H thiab 1-C; 7-H thiab 9-C; 9-H thiab 1-C, 8-C; {{112} }H2 thiab 7-C, 8-C, 9-C; 1 -H thiab 10-C). Tus txheeb ze stereostructure ntawm 2 yog tus yam ntxwv los ntawm ib theem-rhiab NOESY kev sim, uas qhia NOE kev sib raug zoo ntawm cov nram qab no proton khub (3a-H thiab 4a-H; 3b-H thiab 4b-H; 4b-H thiab 5- H; 5-H thiab 6b-H, 9-H) raws li qhia nyob rau hauv daim duab 1. Yog li, lub stereostructure ntawm 2 tau elucidated raws li qhia.

Kankanoside N (3) raug cais raws li cov hmoov dawb uas muaj qhov tsis zoo ntawm kev sib hloov kho qhov muag ([a]D 25 24.6 hauv MeOH). Hauv qhov zoo-ion FAB-MS ntawm 3, lub ncov quasimolecular ion tau pom ntawm m / z 371 [M Na] . Cov mis mos molecular C16H28O8 tau txiav txim siab los ntawm high-resolution FAB-MS ntsuas. Acid hydrolysis ntawm 3 nrog 1.0 M HCl liberated D-glucose. Cov ntaub ntawv 1 H- thiab 13C-NMR (CD3OD, Tables 1, 2) pom cov cim qhia tau rau methyl [d 1.07 (3H, d, J 7.2 Hz, 10-H3)] , plaub methylene {d 1.37, 1.87 (1H txhua, ob qho tib si m, 7-H2), 1.65, 1.81 (1H txhua, ob qho tib si m, 6-H2), [3.65 (1H, dd, J 9.1) , 9.8 Hz), 3.90 (1H, dd, J 5.9, 9.8 Hz), 11-H2], thiab [3.70 (1H, dd, J 3.3, 12.0 Hz), 3.88 (1H, m), {{ 71}}H2]}, plaub methines [d 1.69 (1H, m, 4-H), 1.75 (1H, m, 9-H), 2.06 (1H, m, 8-} H), 2.16 (1H, m, 5-H)], thiab ib pawg hemiacetal [d 4.67 (1H, d, J 7.4 Hz, 1-H)] ua ke nrog bD-glucopyranosyl moiety [ d 4.26 (1H, d, J 7.9 Hz, 1 -H)]. Cov qauv iridoid ntawm 3 tau qhia meej los ntawm 1 H–1 H COZY thiab HMBC cov kev sim thiab cov txheeb ze stereostructure yog tus cwj pwm los ntawm kev sim theem-rhiab NOESY raws li qhia hauv daim duab 1. Yog li ntawd, sterostructure ntawm 3 tau elucidated raws li qhia.

Cistanche tubulosa

Cov qauv ntawm Kankanosides O (4) thiab P (5)

Kankanosides O (4) thiab P (5), C16H26O8, kuj tau txais cov hmoov dawb nrog kev sib hloov kho qhov muag tsis zoo (4: [a]D 23 26.1; 5: [a]D 21 32.7 ob leeg hauv MeOH). IR spectra ntawm 4 thiab 5 tau pom kev nqus bands ntawm 3433, 1696, 1647, thiab 1076 cm 1 rau 4, thiab ntawm 3434, 1701, 1647, thiab 1{{96} 76 cm 1 rau 5, ascribable rau glycosidic, carboxyl, thiab olefin zog. Lawv cov UV spectra tau pom qhov kev nqus siab tshaj plaws ntawm 217 nm qhia tias muaj b-unsaturated carboxylic acid moiety hauv ob qho tib si. Acid hydrolysis ntawm 4 thiab 5 liberated D-glucose, whereas los ntawm enzymatic hydrolysis nrog b glucosidase, 4 thiab 5 muab (2E,6E)-8-hydroxy-2,6-dimethyl{39 }}, {{40}}octadecenoic acid20) (4a) thiab (2E,6E)-8-hydroxy-3,7- dimethyl{{ 48}}, 6-octadecenoic acid21) (5a), feem. 1 H- thiab 13C-NMR cov ntaub ntawv ntawm 4 (CD3OD, Tables 2, 3) tau pom cov cim qhia tau rau ob lub methyls [d 1.71 (3H, br s, 10-}H3), 1.81 (3H, d, J 1.0 Hz , 9-H3)], peb methylene {d 2.19 (2H, br t, J ca. 7 Hz, 5-H2), 2.36 (2H, m, 4-H2), [ 4.24 (1H, dd, J 7.6, 12.0 Hz), 4.33 (1H, dd, J 6.2, 12.0 Hz), 8-H2]}, thiab ob trisubstituted olefins [d 5.41 (1H, ddd, J 1.2) 6.2, 7.6 Hz, 7- H), 6.75 (1H, tq, J 7.2, 1.0 Hz, 3-H)] ua ke nrog bD glucopyranosyl ib feem [d 4.34 (d, J 7.8 Hz, 1 -H)]. Los ntawm kev sib piv ntawm cov paib carbon nyob rau hauv 13C-NMR spectrum ntawm 4 nrog cov ntawm 4a, glycosylation hloov pauv tau pom ntawm 8- txoj hauj lwm (d C 4: 65.5; 4a: 59.4). Txoj hauj lwm ntawm kev sib txuas ntawm glucoside kuj tau lees paub los ntawm HMBC cov kev sim raws li qhia hauv daim duab 2. Yog li ntawd, stereostructure ntawm 4 tau qhia meej tias yog (2E,6E)-8-bD-glucopyranosyloxy-2,{{ 143}}dimethyl{144}}, 6-octadecenoic acid. Ntawm qhov tod tes, 1 H- thiab 13C-NMR cov ntaub ntawv ntawm 5 (CD3OD, Tables 2, 3) qhia tias muaj ib (2E,6E)-8-hydroxy-3,{{158} }dimethyl{159}},6-octadecenoic acid moiety [d 1.70 (3H, br s, 10-H3), 2.14 (3H, br s, 9-H3), 2.24 ( 2H, m, 4-H2), 2.27 (2H, m, 5-H2), 4.05, 4.20 (1H txhua, ob qho tib si br d, J ca. 12 Hz, 8-H2) , 5.47 (1H, tq, J 7.1, 0.9 Hz, 6-H), 5.67 (1H, br s, 1-H)] ua ke nrog bD-glucopyranosyl part [d 4.23 (d, J 7.7 Hz, 1 -H)]. Kev sib txuas ntawm bD-glucopyranosyl moiety nyob rau hauv 5 yog elucidated nyob rau hauv lub hauv paus ntawm HMBC thwmsim raws li qhia nyob rau hauv daim duab 2. Tsis tas li ntawd, ib tug raug glycosylation hloov tau raug soj ntsuam rau cov teeb liab ntawm 8- txoj hauj lwm (d C 5: 75.6; 5 a:68,8 ib. Raws li cov ntaub ntawv pov thawj saum toj no, lub stereostructure ntawm 5 tau txiav txim siab tias yog (2E,6E)-8-bD-glucopyranosyloxy-3,7-dimethyl-2,{ {230}}octa dienoic acid.

Cov txiaj ntsig ntawm Cov Khoom Siv Rau Cov Mob Necrosis Factor-a (TNF-a)-vim Cytotoxicity hauv L929 Cells

TNF-a tau paub los kho ntau yam kev raug mob hauv nruab nrog cev los ntawm nws qhov kev cuam tshuam ntawm cellular apoptosis. Nyob rau hauv cov ntaub ntawv ntawm lub siab, cov teebmeem lom ntawm TNF-a tau cuam tshuam rau kev raug mob ntawm lub siab vim yog cov co toxins, ischemia / reperfusion, viral hepatitis, thiab cawv.22-24) Yog li, TNF-a yog suav tias yog ib qho tseem ceeb. Lub hom phiaj los nrhiav cov tshuaj tiv thaiv kab mob thiab hepatoprotective. Raws li lub tswv yim hais saum toj no, peb tau tshawb xyuas cov khoom tiv thaiv los ntawm cov khoom tshwm sim ntawm TNF-a-induced cell tuag nyob rau hauv L929 hlwb, TNF-a-sensitive cell kab.25) Yav dhau los, peb tau tshaj tawm tias ntau cov khoom siv los ntawm Piper Chaba, 26–29) Boesenbergia rotunda, 30,31) Punica granatum, 32) Helichrysum arenarium, 33–35) thiab Sapindus rarak, 36–38) tau pom muaj nyob rau hauv hibitory teebmeem ntawm TNF-a -induced cytotoxicity hauv L929 hlwb. Txij li thaum cov phenylethanoid constituents ntawm C. tubulosa (xws li echinacoside, acteoside, thiab isoacteoside, thiab lwm yam) 5) kuj inhibited no cytotoxicity, peb ntxiv tshuaj iridoid, phenylpropanoid, thiab lignan constituents raws li qhia nyob rau hauv Table 4. Raws li qhov tshwm sim, kankanoside. (9, inhibition: 16.3 2.0 feem pua ​​​​ntawm 100 mM), mussaenosidic acid (10, 44.7 8.7%), 8-epiloganic acid (11, 10.7 0.4%), 8-hydroxygeraniol 8-ObD-glucopyranoside (17, 21.3 2.4%), thiab ( )-pinoresinol ObD-glucopyranoside (22, 22.3 1.6%), tau pom tias muaj kev ua haujlwm tsis zoo. Txawm hais tias lawv cov dej num tsis muaj zog dua li cov echinacoside (IC50 31.1 mM), acteoside (17.8 mM), thiab isoaceteoside (22.7 mM), lub hauv paus ntsiab lus phenylethanoid constituents.5)

Kev sim

Cov cuab yeej hauv qab no tau siv los muab cov ntaub ntawv spectral thiab lub cev: kev sib hloov tshwj xeeb, Horiba SEPA-300 digital polarimeter (l 5 cm); UV spectra, Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrometer; 1 H- thiab 13C-NMR spectra, JEOL JNM-ECA600 (600, 150 MHz) thiab JEOL JNM-ECS400 (400, 100 HMz) spectrometers nrog tetramethylsilane raws li tus qauv sab hauv; FAB-MS thiab high-resolution FAB-MS, JEOL JMS-SX 102A huab hwm coj spectrometer; HPLC detector, Shimadzu RID-10A refractive index, Shimadzu SPD-10A UV–VIS, thiab Shodex OR-2 optical rotation detectors. HPLC kem, Cosmosil 5C18-MS-II, thiab pNAP (Nacalai Tesque Inc., 250 4.6 mm id) thiab (250 20 mm id) kab tau siv rau kev tshuaj xyuas thiab kev npaj, raws li.

Cov kev sim hauv qab no tau siv rau chromatography: normal-theem silica gel column chromatography (CC), silica gel 60N (Kanto Chemical Co., Ltd., 63-210 mesh, spherical, nruab nrab); reversed-phase silica gel CC, Diaion HP-20 (Nippon Rensui), thiab Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100-200 mesh); ib txwm-theem TLC, pre-coated TLC daim hlau nrog silica gel 60F254 (Merck, 0.25 hli); thim rov qab-theem TLC, pre-coated TLC daim hlau nrog silica gel RP-18 F254S (Merck, 0.25 mm); thim rov qab-theem HPTLC, pre-coated TLC daim hlau nrog silica gel RP-18 WF254S (Merck, 0.25 mm), qhov kev kuaj pom tau tiav los ntawm kev txau nrog 1 feem pua ​​Ce (SO4) 2- 10 feem pua ​​aqueous H2SO4, ua raws li cov cua sov .

Cov khoom cog

Cov khoom no tau piav qhia hauv tsab ntawv tshaj tawm dhau los.

Extraction thiab rho tawm

Cov stems tshiab ntawm C. tubulosa (2.98 kg) raug txiav kom zoo thiab muab rho tawm peb zaug nrog methanol hauv qab reflux rau 3 h. evaporation ntawm cov kuab tshuaj nyob rau hauv txo siab muab ib tug methanolic extract (249.1 g, 8.36 feem pua). Cov tshuaj methanolic tau raug rau Diaion HP-20 CC (5.0 kg, H2O→MeOH) muab H2O- thiab MeOH-eluted fractions (167.84 g, 5.63 feem pua ​​thiab 81.21 g, 2.73 feem pua, raws). Lub MeOH-eluted frac tion (61.00 g) raug rau cov theem ib txwm muaj silica gel CC [1.8 kg, CHCl3– MeOH–H2O (15 : 3 : 0.4→1{{ 36}} : 3 : 0.5→6 : 4 : 1, v/v/v) →MeOH] muab xya feem [Fr. 1 (1.12 g), 2 (9.56 g), 3 ({76}}.89 g), 4 (10.69 g), 5 (8.84 g), 6 (12.52 g), thiab 7 (4.6{{104}} g)], raws li tau piav qhia yav dhau los.5) Cov feem 1 (1.12 g) tau sib cais los ntawm kev thim rov qab-theem silica gel CC [55 g, MeOH–H2O (4 0 : 60, v/v) →MeOH] muab tsib feem [Fr. 1-1 (132.8 mg), 1-2 (267.0 mg), 1-3 (182.8 mg), {{80}} (31{{ 156}}.9 mg), thiab 1-5 (91.4 mg)]. Cov feem 1-1 (132.8 mg) tau ntxiv purified los ntawm HPLC [Cosmosil 5C{{9{160}}}}MS-II, CH3CN–1 feem pua ​​aqueous AcOH (1{{166}) } : 90, v/v)] muab kankanoside M (2, 2.8 mg, 0}.{{203}}{{22{24{248} }}}}}}01% ), bartsioside (7, 5.2 mg, 0). }}6}}O- bD-glucopyranoside (21, 1.4 mg, {{3{{3{3{31{315}}}}9}}5}}{{320} }}} 0001% ). Cov feem 1-2 (267.{{350}} mg) tau ntxiv purified los ntawm HPLC [Cosmosil 5C18-MS-II, CH3CN–1 feem pua ​​aqueous AcOH (7 : 93, v/v)] muab (2E,6E){127}},7-dimethyl-8-hydroxyoctadien{{13{{360}}}}yl-O-bD -glucopyranoside (16, 15.8 mg, {{370}}}. 8-ObD glucopyranoside (17, 5.2 mg, 0.0002%), ( )-pinoresinol ObD-glucopyra noside (22, 22.0 mg, 0.0010%) , eucommin A (23, 5.0 mg, 0.0002%) (24, 21.3 mg, 0.0010%), thiab ( )-syringaresinol ObD-glucopy ranoside (25, 98.0 mg, 0.0044%). Qhov feem 2 (9.56 g) tau sib cais los ntawm kev thim rov qab-theem silica gel CC [400 g, MeOH–H2O (10 : 90, v/v) → MeOH → acetone] muab tsib feem [Fr. 2-1 (55.9 mg), 2-2 (4.48 g), 2-3 (3.42 g), 2-4 (1.16 g), thiab 2-5 (31.9 mg) ], raws li tau piav qhia yav dhau los.5) Qhov feem 2-2 (500.0 mg) raug rau HPLC [Cosmosil 5C18-MS-II, CH3CN–1 feem pua ​​​​aqueous AcOH (5 : 95, v/v )] muab kankanoside L (1, 6.6 mg, 0.0026%) thiab 6-deoxycatalpol (6, 455.5 mg, 0.182%). Cov feem 2-3 (1.00 g) raug muab tso rau HPLC [Cosmosil 5C18-MS-II, CH3CN–1 feem pua ​​aqueous AcOH (7 : 93, v/v)] muab xya feem [Fr . 2-3-1 (66.5 mg), 2-3-2 (20.4 mg), 2-3-3 (26.0 mg), 2-3-4 (136.6 mg), 2-3-5 [7 (380.6 mg), , 0.0581%)], 2-3-6 [ gluroside (8, 285.1 mg, 0.0435%)], thiab 2-3-7 (84.9 mg)]. Cov feem 2-3-4 (106.6 mg) tau ntxiv purified los ntawm HPLC [Cosmosil pNAP, CH3CN–1 feem pua ​​aqueous AcOH (5 : 95, v/v)] muab 6 (68.4 mg, 0.0134 feem pua) ua ke nrog salidroside5) (13.7 mg, 0.0027 feem pua). Cov feem 2-4 (1.16 g) raug rau HPLC [Cosmosil 5C18-MS-II, CH3CN–1 feem pua ​​aqueous AcOH (15 : 85, v/v) thiab Cosmosil pNAP, CH3CN–1 feem pua Aqueous AcOH (10 : 90 lossis 15 : 85, v/v)] muab kankanosides N (3, 15.6 mg, 0.0007%), O (4, 44.1 mg, 0.0020%), thiab P (5, 4.2 mg, 0.0002) 6 (24.0 mg, 0.0011%), 8 (17.7 mg, 0.0008 feem pua), kankanoiside A (9, 22.3 mg, 0.0010%), 8-epideoxyloganic acid (12, 8.1 mg, 0.004%) kankanoside E (14, 58.1 mg, 0.0026%), (2E,6Z)-8-bD-glucopy ranosyloxy-2,6-dimethyl-2,6-octadienoic acid (15, 31.0 mg, 0.0014%), 16 (168.6 mg, 0.0075 feem pua), 17 (94.3 mg, 0.0042%), betualbuside A (18, 8.1 mg, 0.0004%), coniferin. ), thiab syringin (20, 33.9 mg, 0.0015 feem pua). Qhov feem 4 (10.69 g) tau sib cais los ntawm kev thim rov qab-theem silica gel CC [500 g, MeOH–H2O (30 : 70, v/v) → MeOH → acetone] muab plaub feem [Fr. 4-1 (878.2 mg), 4-2 (7.06 g), 4-3 (1.57 g), thiab 4-4 (792.8 mg)], raws li tau piav ua ntej.5) Cov feem 4-1 (500.0 mg) yog purified los ntawm HPLC [Cosmosil 5C18-MS-II, CH3CN–1 feem pua ​​aqueous AcOH (10 : 90, v/v)] muab mussaenosidic acid (10, 39.1) mg, 0.0031 feem pua) thiab geniposidic acid (13, 51.2 mg, 0.0040 feem pua). Qhov feem 5 (8.84 g) tau muab cais los ntawm kev thim rov qab-theem silica gel CC [400 g, MeOH–H2O (20 : 80 → 30 : 70, v / v) → MeOH → acetone] muab xya feem [Fr. 5-1 ​​(870.2 mg), 5-2 (478.9 mg), 5-3 (3.72 g), 5-4 (979.9 mg), 5-5 (1.19 g), 5-6 (1.27 g), thiab 5-7 (130.1 mg)], raws li tau piav qhia yav dhau los.5) Cov feem 5-1 (870.2 mg) tau ua kom huv ntxiv los ntawm HPLC [Cosmosil pNAP, CH3CN -1 feem pua ​​aqueous AcOH (5 : 95, v/v)] muab 10 (55.3 mg, 0.0025 feem pua) thiab 8-epiloganic acid (11, 51.5 mg, 0.0023%).

Kankanoside L (1): Cov hmoov dawb, [a]D 26 45.7 (c 0.48, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C15H24O9Na [M Na] 371.1318; Pom 371.1309. IR (KBr, cm 1): 3433, 2928, 1080. 1 H-NMR (600 MHz, CD3OD) d: muab hauv Table 1. 13C-NMR (150 MHz, CD3OD) d C: muab hauv Table 2. Positive- ion FAB-MS m/z: 371 [M Na] . Negative-ion FAB-MS m/z: 347 [MH] .

Kankanoside M (2): Cov hmoov dawb, [a]D 26 18.7 (c 0.11, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C15H22O8Na [M Na] 353.1212; pom 353.1208. IR (KBr, cm 1): 3433, 2924, 1736, 1655, 1076. 1 H-NMR (600 MHz, CD3OD) d: muab hauv Table 1. 13C-NMR (150 MHz, CD3OD) d C: muab rau hauv Table 2. Positive-ion FAB-MS m/z: 353 [M Na] .

Kankanoside N (3): Cov hmoov dawb, [a]D 25 24.6 (c 1.00, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C16H28O8Na [M Na] 371.1682; Pom 371.1686. IR (KBr, cm 1): 3415, 2874, 1076, 1028. 1 H- NMR (600 MHz, CD3OD) d: muab hauv Table 1. 13C-NMR (150 MHz, CD3OD) d C: muab hauv Table 2. Positive-ion FAB-MS m/z: 371 [M Na] .

Kankanoside O (4): Cov hmoov dawb, [a]D 23 26.1 (c 2.30, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C16H26O8Na [M Na] 369.1525; pom 369.1528. UV [l max (log e), MeOH, nm]: 217 (4.04). IR (KBr, cm 1): 3433, 2926, 1696, 1647, 1076. 1 H-NMR (600 MHz, CD3OD) d: muab hauv Table 3. 13C-NMR (150 MHz, CD3OD) d C: muab rau hauv Table 2. Positive-ion FAB-MS m/z: 369 [M Na] .

Kankanoside P (5): Cov hmoov dawb, [a]D 21 32.7 (c 0.18, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C16H26O8Na [M Na] 369.1525; pom 369.1520. UV [l max (log e), MeOH, nm]: 217 (4.08). IR (KBr, cm 1): 3434, 2926, 1701, 1647, 1076. 1 H-NMR (600 MHz, CD3OD) d: muab hauv Table 3. 13C-NMR (150 MHz, CD3OD) d C: muab rau hauv Table 2. Positive-ion FAB-MS m/z: 369 [M Na] .

Acid Hydrolysis ntawm Kankanosides L-P (1-5): Kev daws ntawm 1-5 (txhua 1.0 mg) hauv 1.0 M HCl (1.0 ml) yog rhuab ntawm 8 0 degree rau 3 h. Tom qab txias lawm, cov tshuaj tiv thaiv sib tov yog neutralized nrog Amberlite IRA-400 (OH daim ntawv), thiab cov resins raug tshem tawm los ntawm kev pom. Tom qab tshem tawm cov kuab tshuaj nyob rau hauv lub siab txo, cov residue raug cais los ntawm Sep-Pak C18 cartridge kem (H2O→MeOH). Lub H2O-eluted feem yog raug rau HPLC tsom xam raws li nram no: HPLC kem, Kaseisorb LC NH2-60-5, 4.6 hli id ​​250 hli (Tokyo Kasei Co., Ltd., Tokyo, Nyiv); kuaj pom, kho qhov muag rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Nyiv); mobile theem, CH3CN–H2O (85: 15, v/v); Tus nqi sib tw 0.8 ml / min]. Kev txheeb xyuas ntawm D-glucose tam sim no nyob rau hauv H2O-eluted feem ntawm 1-5 tau ua los ntawm kev sib piv ntawm lawv lub sijhawm khaws cia thiab kev hloov kho qhov muag nrog cov qauv qhabnias [tR 17.9 min (zoo)].

Hydrogenation ntawm {{0}}Deoxycatalpol (6) Kev ncua ntawm 6 (12.0 mg) thiab 10 feem pua ​​palladium carbon (5.0 mg) hauv MeOH (2.0 ml) tau nplawm ntawm chav tsev kub hauv qhov chaw H2 rau 2 teev. Cov catalyst tau lim tawm, thiab cov filtrate tau condensed nyob rau hauv lub siab txo kom muab 1 (12.0 mg, quant.).

Enzymatic Hydrolysis ntawm Kankanosides O (4) thiab P (5) nrog b-Glucosidase Rau kev daws ntawm 4 (8.0 mg) hauv H2O (1.5 ml) tau ntxiv b-glucosidase (4.7 mg, los ntawm almond, Oriental Poov Co., Tokyo, Nyiv), thiab cov tshuaj tau nplawm ntawm 37 degree rau 24 teev. Cov tshuaj tiv thaiv tau quenched los ntawm qhov sib ntxiv ntawm EtOH (5.0 ml), qhov sib tov yog condensed nyob rau hauv txo siab. Cov residue tau muab rho tawm nrog EtOAc thiab evaporation ntawm cov kuab tshuaj muab (2E,6E)-8-hydroxy-2,{{20}}dimethyl-2,{{22} octadecenoic acid20) (4a, 3.9 mg, 92 feem pua). Los ntawm cov txheej txheem zoo sib xws, (2E,6E)-8-hydroxy-3,7-dimethyl-2,6-octadecenoic acid21) (5a, 0.9 mg, 94 feem pua ) tau txais los ntawm kankanoside P (5, 1.8 mg).

Bioassay Method Inhibitory nyhuv ntawm TNF-a-induced cytotoxicity hauv L929 hlwb raug soj ntsuam los ntawm txoj kev tau piav qhia hauv daim ntawv dhau los.5)

Statistics Values ​​yog qhia raws li txhais tau tias SEM Ib-txoj kev tsom xam ntawm variance (ANOVA) ua raws li los ntawm Dunnett qhov kev xeem tau siv rau kev txheeb xyuas. Qhov tshwm sim (p) qhov tseem ceeb tsawg dua 0.05 tau suav tias yog qhov tseem ceeb.

cistanche tshuaj ntsuab