Monoterpene Constituents Los ntawm Cistanche Tubulosa-Chemical Structures Ntawm Kankanosides A-E Thiab Kankanol-

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

Haihui XIE, Toshio MORIKAWA, Hisashi MATSUDA, Seikou NAKAMURA, Osamu MURAOKA, thiab Masayuki YOSHIKAWA

Abstract

Plaub tshiab iridoid glycosides, kankanosides A (1), B (2), C (3), thiab D (4), chlorinated iridoid, kankanol (5), thiab ib qho acyclic monoterpene glycoside, kankanoside E (6), raug cais tawm. los ntawm methanolic extract ntawm qhuav stems ntawm Cistanche tubulosa (SCHRENK) R. WIGHT (Orobanchaceae) ua ke nrog 16 paub tebchaw. Cov qauv ntawm cov tebchaw tshiab no (1-6) tau txiav txim siab raws li cov pov thawj tshuaj lom neeg thiab physicochemical.

Lo lus tseem ceeb:Cistanche tubulosa; kankanoside; kankanol; iridoid; monoterpene; Orobanchaceae

Cistanche tubulosa(SCHRENK) R. WIGHT (Orobanchaceae)yog ib tsob nroj uas muaj kab mob perennial loj hlob ntawm cov hauv paus hniav ntawm Salvadora lossis Calotropis hom, thiab muab faib rau hauv North Africa, Arabia, thiab Asian lub tebchaws. , lumbago, thiab lub cev tsis muaj zog.2) Yav tas los, ob peb iridoids, monoterpenoids, phenylethanoids, thiab lignans raug cais los ntawm Suav thiab Pakistan C. tubulosa. 1,3–7) Hauv chav kawm ntawm peb cov kev tshawb fawb serial ntawm bioactive constituents los ntawm Suav natural tshuaj, 8–18) plaub tshiab iridoid glycosides, kankanosides A (1), B (2), C (3), thiab D (4 ), chlorinated iridoid, kankanol (5), thiab ib qho acyclic monoterpene glycoside, kankanoside E (6), raug cais tawm los ntawm cov tshuaj methanolic ntawm cov tshuaj ntsuab no ua ke nrog 16 lub tebchaw paub suav nrog 12 monoterpenes (7-18). Daim ntawv no hais txog kev sib cais thiab cov qauv elucidation ntawm cov tshiab monoterpene constituents (1-6).

Cistanche tubulosa

Methanol extract los ntawm qhuav stems ntawmCistanche tubulosa(26.8 feem pua ​​​​ntawm cov tshuaj ntsuab no) tau raug rau theem ib txwm thiab thim rov qab-theem silica gel kem chromatography thiab rov HPLC kom muab kankanosides A (1, 0). {7}}{{10 }}54% ), B(2, 0). 27 feem pua ​​), thiab D (4, {{40}} . {66}}34% ), thiab kankanoside E (6, 0.027 feem pua ​​) ua ke nrog mussaenosidic acid19) (7, 0.020 feem pua ​​) , geniposidic acid19) (8, 0.030%), 8-epiloganic acid7) (9, 0.033 feem pua), gluroside19) (10, 0.14%), antirrhide20) (11, 0.0079 feem pua), ajugol, 10. feem pua ​​), bartsioside19) (13, 0.21%), 6-deoxycatalpol19) (14, 0.11 feem pua), argyol21) (15, 0.0030 feem pua), cistanin22,23) (16, 0.0040 feem pua), cis22 (cis) 17, 0.0035% ), (2E,6Z)-8-bD-glucopyranosyloxy-2,6-dimethyl-2,6-}octadienoic acid24) (18, 0.0028%) ), D mannitol25) (4.19 p ercent ), uridine25) (0.0069%), (3R)-3-hydroxy-2- pyrrolidinone26,27) (0.0020%), thiab (3R)-3-hydroxy-1-methyl{ {97}}pyrrolidinone27) (0.0059 feem pua ​​).

Cov qauv ntawm Kankanoside A (1) Kankanoside A (1) tau txais los ua cov hmoov amorphous thiab nthuav tawm qhov kev sib hloov tsis zoo ([a]D 25 107.4 degree hauv MeOH). IR spectrum ntawm 1 tau pom ib qho nqus nqus ntawm 1647 cm 1 tau muab rau olefin muaj nuj nqi ntxiv rau cov khoom nqus nqus ntawm 3410 thiab 1076 cm 1 qhia txog glycoside moiety. Nyob rau hauv qhov zoo- thiab tsis zoo-ion ceev atom bombardment (FAB)-MS ntawm 1, quasimolecular ion peaks tau pom ntawm m / z 369 (M Na) thiab 345 (MH), thiab high-resolution FAB-MS tsom xam qhia cov molecular Cov mis mos ntawm 1 yog C16H26O8. 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 ntsuas qhov muag.8,10-12,15-18) Lub 1 H- (CD3OD, Table 1) thiab 13C-NMR (Table 2) spectra ntawm 1, uas tau muab los ntawm ntau yam kev sim NMR, 28) pom cov cim qhia tau rau ob lub methyls [d 1.31 (s, 10-H3), 1.51 (br s, {{47}) }H3)], ob methylene [d 1.49, 2.02 (ob leeg m, 6a- thiab 6b-H), 1.64, 1.67 (ob leeg m, 7b- thiab 7a-H)], ob methines [d 2.21 (dd, J 2.7) , 9.5 Hz, 9-H), 2.71 (m, 5-H)], thiab a, b-unsaturated acetal pawg [d 5.33 (d, J 2.7 Hz, 1-H ), 5.95 (br s, 3-H)] ua ke nrog b-glucopyranosyl moiety [d 4.62 (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 correlations (HMBC) ntawm 1, kev sib raug zoo ntev tau pom ntawm cov protons thiab carbons hauv qab no (3-H, 1 -H thiab 1-C; 11- H3 thiab 3-C; 3-H, 5-H, 6-H2, 9-H, 11-H3 thiab 4- C ; 11-H3 thiab 5-C; 10-H3 thiab 7-C; 1-H, 7-H2, 9-H, 10-H3, thiab 8-C; 10-H3 thiab 9-C; 7-H2 thiab 10-C) raws li qhia hauv daim duab 1. Enzymatic hydrolysis ntawm 1 nrog b-glucosidase muab ib qho aglycon, kankagenin a (1a) raws li qhia hauv daim duab 3. Kev sib piv ntawm 13C NMR spectrum rau 1 nrog rau cov 1a qhia txog kev hloov glycosylation nyob ib ncig ntawm 1- txoj hauj lwm hauv 1 [1: dC 94.1 ({147}}C), 134.6 (3-C), 53.3 (9-C); 1a: dC 92.9 ({157}}C), 135.3 (3-C), 54.7 ({163}}C)]. Yog li, kev sib txuas ntawm bD-glucopyranosyl moiety hauv 1 kuj tau qhia meej tias nyob ntawm 1- txoj hauj lwm ntawm 1a. Tom ntej no, tus txheeb ze stereostructure ntawm 1 yog tus cwj pwm los ntawm nuclear Overhauser enhancement spectroscopy (NOESY) kev sim, uas qhia NOE kev sib raug zoo ntawm cov nram qab no proton khub (1-H thiab 10-H3; 3-H thiab 11-H3; 5-H thiab 6b-H, 9-H; 6b-H thiab 7b-H; 7a-H thiab 10-H3; 7b-H thiab {{ 190}}H) raws li qhia hauv daim duab 2. Thaum kawg, qhov kev teeb tsa kiag li ntawm 1- txoj haujlwm hauv 1 tau txiav txim siab los ntawm kev siv 13C NMR glycosylation hloov txoj cai ntawm 1,1 -disaccharide,29) uas tau pom siv rau cov hemiacetal compounds.30,31) Lub stereostructure ntawm lub 1- txoj hauj lwm nyob rau hauv 1 tau lees paub tias yuav tsum tau khaws cia nyob rau hauv 1a los ntawm kev sib piv ntawm 1 H- NMR tsom xam nrog rau cov kev sim NOESY. Kev hloov pauv glycosylation qhov tseem ceeb [Dd 1.2 ppm (1 -C) thiab 0.9 ppm (1- C), hauv pyridine-d5] tau pom tias yog tus yam ntxwv ntawm R, Rhemiacetal ua ke, uas sib haum rau qhov tsis muaj stereostructure ntawm 1. raws li qhia nyob rau hauv daim duab 3. Yog li ntawd, lub meej configuration ntawm lub 1- txoj hauj lwm ntawm 1 tau txiav txim siab los ua S configuration, thiab lub meej stereostructure ntawm 1 yog elucidated raws li qhia.

Cov qauv ntawm Kankanosides B (2) thiab C (3) Kankanoside B (2) kuj raug cais tawm raws li cov hmoov amorphous nrog qhov tsis zoo ntawm kev sib hloov kho qhov muag ([a]D 26 118.7 degree hauv MeOH). IR spectrum ntawm 2 tau pom cov khoom nqus nqus ntawm 3410, 1647, thiab 1{{5{{8{90}}}}}}}85 cm 1 ascribable rau hydroxyl, olefin, thiab ether functions. Cov mis mos molecular C15H24O10 ntawm 2 tau txiav txim siab los ntawm quasimolecular ion peaks hauv qhov zoo-ion FAB-MS thiab los ntawm high-resolution FAB-MS. Acid hydrolysis ntawm 2 nrog 1.0 M HCl liberated D-glucose, uas tau txheeb xyuas los ntawm HPLC kev tshuaj xyuas siv lub ntsuas ntsuas qhov muag.8,10-12,15-18) Lub 1 H- (CD3OD, Table 1) thiab 13C-NMR ( Table 2) spectra28) ntawm 2 pom cov cim qhia tau rau ob lub methylene [d 1.40 (DDD, J 5.2, 7.3, 13.5 Hz, 6a-H), 2.52 (DDD, J 7.0, 9.2, 13.5 Hz, 6b-H), 3. , 3.99 (ob leeg d, J 11.9 Hz, 10-H2)], plaub methines [d 2.21 (dd, J 6.4, 8.6 Hz, 9-H), 2.83 (m, 5-} H), 4.02 (dd, J 5.2, 7.0 Hz, 7-H), 5.49 (d, J 6.4 Hz, 1-H)], thiab ib khub cisolefifin [d 4.95 (dd, J 4.0) , 6.1 Hz, 4-H), 6.22 (dd, J 1.8, 6.1 Hz, 3-H)], ua ke nrog b-glucopyranosyl moiety [d 4.72 (d, J 7.9 Hz, 1 - H)]. Cov paib proton thiab carbon nyob rau hauv 1 H- thiab 13C-NMR cov ntaub ntawv ntawm 2 tau zoo ib yam li cov ntawm 6-deoxycatalpol (14), tshwj tsis yog rau cov teeb liab vim yog 7- thiab 8- txoj haujlwm. Raws li pom nyob rau hauv daim duab 1, 1 H–1 H COZY kev sim ntawm 2 qhia tias muaj ib feem ntawm cov qauv sau nyob rau hauv bold kab thiab, nyob rau hauv lub HMBC kev sim, ntev-ntev correlations tau soj ntsuam ntawm cov nram qab no proton thiab carbon khub ({{ 122}}H, 1 -H thiab 1-C; 1-H thiab 3-C; 10-H2 thiab 7- C; 1-H , 7-H, 9-H, 10-H2, thiab 8-C; 7-H, 10-H2 thiab 9-C ; 7-H thiab 10-C). Tus txheeb ze stereostructure ntawm 2 yog tus cwj pwm los ntawm qhov kev sim NOESY, uas qhia NOE kev sib raug zoo ntawm cov proton khub hauv qab no (1-H thiab 10-H2; 3- H thiab 4-H; 5-H thiab 6b-H, 9-H; 6b-H thiab 7-H; 7-H thiab 9-H) raws li qhia hauv daim duab 2. Thaum kawg, alkaline kev kho mob ntawm 14 nrog 5 feem pua ​​aqueous potassium hydroxide (KOH) yielded 2 thiab 19, 32) kom lub stereostructure ntawm 2 tau qhia meej.

Kankanoside C (3) raug cais raws li cov hmoov amorphous nrog qhov tsis zoo ntawm qhov kev sib hloov kho qhov muag ([a]D 26 34.0 degree hauv MeOH). Hauv qhov tsis zoo-ion FAB-MS ntawm 3, ib khub ntawm isotope quasimolecular ion peaks tau pom ntawm m/z 399 thiab 401 (MH). Cov mis mos molecular ntawm 3 tau txiav txim siab yog C15H25ClO10 los ntawm kev ntsuas siab FAB-MS. Acid hydrolysis ntawm 3 nrog 1.0 M HCl liberated D-glucose, uas tau txheeb xyuas los ntawm HPLC kev tshuaj xyuas siv lub ntsuas ntsuas qhov muag.8,10-12,15-18) Lub 1 H- (CD3OD, Table 1) thiab 13C-NMR ( Table 2) spectra28) ntawm 3 pom cov cim qhia tau rau peb methylene [d 1.70 (br dd, J ca. 3, 13 Hz, 4a-H), 2.70 (br dd, J ca. 6, 13 Hz, 4b-H) , 1.68 (br d, J ca. 13 Hz, 6a H), 2.44 (m, 6b-H), 4.01, 4.04 (ob leeg d, J 11.3 Hz, 10-}H2)], tsib methines [d 2.47 (dd, J 2.5, 7.9 Hz, 9-H), 2.61 (m, 5- H), 3.94 (br s, 7-H), 5.10 (br d, J ca. 3 Hz, 3-H), 5.48 (d, J 2.5 Hz, 1-H)], thiab b-glucopyranosyl moiety [d 4.60 (d, J 8.0 Hz, 1 -H)]. Cov paib proton thiab carbon nyob rau hauv 1 H- thiab 13C-NMR spectra ntawm 3 yog superimposable ntawm 2, tshwj tsis yog rau cov teeb liab vim yog 3- thiab 4- txoj hauj lwm. Cov hauj lwm ntawm bD-glucopyranosyl thiab chlorine muaj nuj nqi hauv 3 tau elucidated los ntawm H-H COZY thiab HMBC thwmsim raws li qhia hauv daim duab 1. Yog li ntawd, cov qauv qauv ntawm 3 tau tsim ua raws li qhia. Tus txheeb ze stereostructure ntawm 3 tau txiav txim siab los ntawm kev sim NOESY, nyob rau hauv uas NOE kev sib raug zoo tau pom ntawm cov nram qab no proton khub (1-H thiab 3-H, 10-H2; 3- H thiab 4a-H; 4b-H thiab 5- H; 5-H thiab 6b-H, 9-H; 6b-H thiab 7-H; {{121} }H thiab 9-H) raws li qhia hauv daim duab 2.

Cov qauv ntawm Kankanoside D (4) thiab Kankanol (5) Kankanoside D (4) raug cais raws li cov hmoov amorphous nrog qhov tsis zoo ntawm qhov kev sib hloov ([a]D 25 30.6 degree hauv MeOH). IR spectrum ntawm 4 tau pom ib qho kev nqus ntawm 1655 cm 1 ascribable rau olefin muaj nuj nqi thiab muaj zog nqus bands ntawm 3410 thiab 1078 cm 1 qhia txog nws cov qauv glycosidic. Hauv qhov zoo-ion FAB-MS ntawm 4, lub ncov quasimolecular ion tau pom ntawm m / z 341 (M Na). Cov mis mos molecular C15H26O7 ntawm 4 tau txiav txim siab los ntawm high-resolution FAB-MS ntsuas. Acid hydrolysis ntawm 4 nrog 1.0 M HCl liberated D-glucose, 8,10-12,15-18) whereas (R)-rotundity (4a) 33,34) tau txais los ntawm enzymatic hydrolysis ntawm 4 nrog b-glucosidase. Lub 1 H-NMR (Table 3, CD3OD) thiab 13C-NMR (Table 4) spectra28) ntawm 4 pom cov cim qhia tau rau methyl [d 1.69 (s, 10-H3)], peb methylene [d 1.41, 2.06 (ob leeg m, 4-H2), 1.51, 2.01 (ob leeg m, 6a- thiab 6b-H), 2.23 (br dd, J ca. 8, 15 Hz, 7a-H), 2.37 (br dd , J ca. 8, 15 Hz, 7b-H)], a methine [d 2.90 (m, 5-H)], thiab ob lub methylene bearing oxygen function {d [3.56 (DDD, J 2.8, 7.4)] , 13.2 Hz), 3.97 (DDD, J 4.9, 8.0, 13.2 Hz), 3-H2], 4.04, 4.18 (ob leeg d, J 12.2 Hz, 1-H2)} ua ke nrog b- glucopyranosyl ntu [d 4.25 (d, J 7.7 Hz, 1 -H)]. Txoj hauj lwm ntawm bD-glucopyranosyl ib feem hauv 4 tau qhia meej los ntawm HMBC kev sim ua 3- txoj haujlwm (Fig. 1). Raws li cov pov thawj no, qhov tseeb stereostructure ntawm 4 tau txiav txim siab ua raws li qhia.

Kankanol (5) tau txais los ua cov hmoov amorphous nrog kev sib hloov kho qhov muag zoo ([a]D 25 11.1 degree ). Cov tshuaj ionization (CI)-MS ntawm 5 tau pom ib khub ntawm isotope ion peaks ntawm m / z 221 thiab 223 vim yog quasimolecular ion (MH). Qhov kev daws teeb meem siab CI-MS ntsuas ntawm 5 qhia cov qauv molecular yog C9H13ClO4. 1 H-NMR (Table 1, CD3OD) thiab 13C-NMR (Table 2) spectra28) ntawm 5 tau pom tias muaj cov haujlwm hauv qab no: peb methylene [d 1.60 (DDD, J 2.5, 5.5, 13.1) Hz, 4a-H), 1.80 (br dd, J ca. 3, 13 Hz, 4b H), 1.83 (br d, J ca. 12 Hz, 6a-H), 2.57 (m, 6b-H), 3.75 , 3.88 (ob leeg d, J 9.2 Hz, 10-H2)], tsib methines [d 2.76 (dd, J 4.3, 8.0 Hz, 9-H), 2.50 (m, 5-} H), 3.80 (br s, 7-H), 5.17 (br d, J ca. 3 Hz, 3-H), 5.26 (d, J 4.3 Hz, 1-H) ]. Cov qauv qauv ntawm 5 tau lees paub los ntawm 1 H–1 H COZY thiab HMBC kev sim. Ntawd yog, qhov kev sim 1 H–1 H COZY ntawm 5 tau qhia tias muaj cov qauv ib nrab sau ua cov kab bold, thiab hauv HMBC kev sim, kev sib raug zoo ntev tau pom raws li qhia hauv daim duab 1. Tus txheeb ze stereostructure ntawm 5 yog. txiav txim los ntawm NOESY kev sim, nyob rau hauv uas NOE kev sib raug zoo tau soj ntsuam ntawm cov nram qab no ing proton khub (1-H thiab 9-H; 3-H thiab 4a-H; 4b-H thiab {{ 93}} H; 5-H thiab 6b-H, 9-H; 6b-H thiab 7-H; 7-H thiab 9-H) raws li qhia nyob rau hauv daim duab 2. Los ntawm kev sib piv ntawm 1 H- thiab 13C-NMR cov ntaub ntawv rau 5 nrog rau cov 14a, uas tau txais los ntawm kev kho mob ntawm 14 nrog 5 feem pua ​​aqueous HCl raws li qhia nyob rau hauv daim duab 1,35) txoj hauj lwm ntawm cov tshuaj chlorine. pab pawg hauv 5 tau txais kev txhawb los ua 3- txoj haujlwm. Tsis tas li ntawd, acetylation ntawm 5 nrog acetic anhydride (Ac2O) thiab pyridine yielded 3, 7-oxide (5a), thaum 14a muab cov diacetate (14b) nyob rau hauv tib yam acetylation mob. Cov pov thawj no kuj tau coj peb kom paub meej tias txoj haujlwm ntawm cov tshuaj chlorine ua haujlwm yog 3b-txoj hauj lwm (Daim duab 3). Yog li ntawd, stereostructure ntawm 5 tau txiav txim siab ua raws li qhia.

Cov qauv ntawm Kankanoside E (6) Kankanoside E (6) raug cais raws li cov hmoov amorphous nrog qhov tsis zoo ntawm qhov kev sib hloov ([a]D 25 20.0 degree hauv MeOH). IR spectrum ntawm 6 tau pom kev nqus bands ntawm 3410, 1647, 1085 cm 1 ascribable rau glycosidic thiab carbonyl zog, thaum nws UV spectrum pom qhov nqus siab tshaj plaws ntawm 211 nm (log e 4.63), qhia tias muaj cov carboxylic acidsaturated. Cov mis mos molecular C16H28O8 ntawm 6 yog tus cwj pwm los ntawm qhov zoo- thiab tsis zoo-ion FAB-MS thiab los ntawm kev ntsuas MS siab. Acid hydrolysis ntawm 6 liberated D-glucose, 8,10-12,15-18) whereas (2E,6R) -8-hydroxy-2, 6-dimethyl- 2-octanoic acid (6a) 36) tau txais los ntawm enzymatic hydrolysis ntawm 6 nrog b-glucosidase. 1 H-NMR (Table 3, CD3OD) thiab 13C-NMR (Table 4) spectra28) ntawm 6 qhia tias muaj ib (2E,6R)-8-hydroxy-2,6- dimethyl-2-octanoic acid moiety [d 0.95 (d, J 6.4 Hz, 10-H3), 1.30, 1.48 (ob leeg m, 5-} H2), 1.45, 1.70 (ob leeg m, { {70}}H2), 1.65 (m, 6-H), 1.81 (s, 9-H3), 2.22 (2H, m, 4-H2), 3.61, 3.94 (ob leeg m, 8-H2), 6.78 (dd, J 1.2, 7.3 Hz, 3-H)] ua ke nrog bD-glucopyranosyl ib feem [d 4.26 (d, J 7.6 Hz, 1 -H)] . Los ntawm kev sib piv ntawm cov paib carbon nyob rau hauv 13C-NMR spectrum ntawm 6 nrog cov ntawm 6a, qhov kev hloov ntawm glycosylation tau pom nyob ib ncig ntawm 8- txoj hauj lwm ntawm 6. Txoj hauj lwm ntawm glucoside linkage kuj tau lees paub los ntawm HMBC kev sim raws li qhia hauv Daim duab 1. Yog li ntawd, qhov tseeb stereostructure ntawm 6 tau qhia meej tias yog (2E,6R)-8-b- D-glucopyranosyloxy-2,6-dimethyl-2-octanoic acid.

cistanche extract txiaj ntsig

Kev sim

Cov cuab yeej hauv qab no tau siv los muab cov ntaub ntawv 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; EI-MS, CI-MS, thiab high-resolution CI-MS, JEOL JMS-GCMATE huab hwm coj spectrometer; FAB-MS thiab high-resolution MS, JEOL JMS-SX 102A huab hwm coj spectrometer; 1 H-NMR spectrum, JEOL EX{15}} (270 MHz) thiab JNM-LA500 (500 MHz) spectrometers; 13C-NMR spectra, JEOL EX-270 (68 MHz) thiab JNM-LA500 (125 MHz) spectrometers nrog tetramethylsilane raws li tus qauv sab hauv; thiab HPLC detector, Shimadzu RID-6A refractive index thiab SPD- 10Avp UV-VIS detectors. HPLC kem, YMC-Pack ODS-A (250 4.6 mm id), thiab (250 20 mm id) kab tau siv rau kev tsom xam thiab kev npaj, feem.

Cov kev sim hauv qab no tau siv rau chromatography: zoo tib yam-theem silica gel kem chromatography, Silica gel BW-200 (Fuji Silysia Chemical, Ltd., Aichi, Nyiv, 15{{10}}-35 0 mesh); rov qab-theem silica gel kem chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., Aichi, Nyiv, 100-200 mesh); TLC, precoated TLC daim hlau nrog Silica gel 60F254 (Merck, 0.25 mm) (ib theem pib) thiab Silica gel RP- 18 F254S (Merck, 0.25 mm) (rov qab theem); thim rov qab-theem HPTLC, precoated TLC daim hlau nrog Silica gel RP-18 WF254S (Merck, 0.25 mm); thiab kev kuaj pom tau tiav los ntawm kev txau nrog 1 feem pua ​​Ce (SO4) 2-10 feem pua ​​aqueous H2SO4 tom qab cua sov.

Cov khoom cog

Qhuav stems ntawm Cistanche tubulosa (SCHRENK) R. WIGHT tau muas ntawm Urumqi, Xinjiang Province, Tuam Tshoj nyob rau hauv Lub ib hlis ntuj 2005 los ntawm Eishin Trading Co., Ltd. Osaka, Nyiv, thiab botanical identification tau ua los ntawm xib fwb Jia Xiaoguang nyob rau hauv Xinjiang lub koom haum ntawm tsoos suav. thiab Ethnologic Medicines. Ib daim ntawv pov thawj (2005.01. Xinjiang-01) ntawm cov nroj tsuag no tau muab tso rau hauv peb chav kuaj.

Extraction thiab rho tawm

Qhuav stems ntawm C. tubulosa (5.0 kg) yog pow dered thiab muab rho tawm peb zaug nrog methanol nyob rau hauv reflux rau 3 h. Evapora tion ntawm cov kuab tshuaj nyob rau hauv lub siab txo tau muab cov methanolic extract (1340 g, 26.8 feem pua ​​ntawm cov tshuaj ntsuab no). Cov tshuaj methanolic extract (160} g) tau raug rau theem ib txwm-theem silica gel kab chromatography [3.2 kg, CHCl3–MeOH–H2O (10} : 3 : 1 → 7 : 3 : 1, qis dua txheej → 6 : 4 : 1) MeOH] muab rau feem ntau [Fr. 1 (5.04 g), Fr. 2 (9.84g), Fr. 3 (7.8{91}} g), Fr. 4 (13.28 g), Fr. 5 (113.6{{104}} g), thiab Fr. 6 (7.61 g). Tshooj 1 (5. 162}} : 50→6{175}} : 40, v/v) →MeOH] them tsib feem [Fr. {{50}} (83{{201}} mg), Fr. 1-2 (590 mg), Fr. 1-3 (180 mg), Fr. 1-4 (124 mg), thiab Fr. 1-5 (3200 mg)]. Fr. {{60}} (830 mg) tau ntxiv los ntawm HPLC [MeOH–H2O (10 : 90, v/v)] muab kankanol (5, 20 mg, 0.0034% ), argyol (15, 18 mg, 0.0030%), cistanin (16, 24 mg, 0.0040%), thiab Fr. 1-1-2 (62 mg), uas tau muab cais ntxiv los ntawm HPLC [MeOH–H2O (2 : 98, v/v)] muab (3R)-3-hydroxy-2-pyrrolidinone (0.0020 feem pua ) thiab (3R)-3-hydroxy-1-methyl-2-pyrrolidinone (0.0059%). Fr. 1-2 (590 mg) tau lim los ntawm HPLC [MeOH–H2O (35 : 65, v/v) thiab CH3CN– H2O (20 : 80, v/v)] muab cistanchlorin (17, 21 mg, 0.0035) feem pua). Tshooj 2 (9.72 g) tau raug thim rov qab-theem silica gel kem chromatography [290 g, MeOH–H2O (20 : 80 → 30 : 70 → 40 : 60 → 60 : 40, v / v) → MeOH] rau afford xya feem [Fr. 2-1 (1986 mg), Fr. 2-2 (1563 mg), Fr. 2-3 (3931 mg), Fr. 2-4 (375 mg), Fr. 2-5 (486 mg), Fr. 2-6 (460 mg), thiab Fr. 2-7 (336 mg)]. Fr. 2-1 (466 mg) raug cais los ntawm HPLC [MeOH–H2O (5 : 95, v/v)] kom muab uridine (0.0069 feem pua). Fr. 2-2 (535 mg) raug cais los ntawm HPLC [MeOH–H2O (10 : 90, v/v)] kom muab tshuaj tua kab mob (11, 15 mg, 0.0079%) thiab 6-deoxycatalpol (14, 214) mg, 0.11 feem pua). Fr. 2-3 (535 mg) tau muab cais los ntawm HPLC [MeOH–H2O (20 : 80, v/v)] los muab gluroside (10, 110 mg, 0.14%) thiab bartsioside (13, 164 mg, 0.21%) . Fr. 2-4 (375 mg) raug cais los ntawm HPLC [MeOH–H2O (30 : 70, v/v)] muab kankanosides A (1, 32 mg, 0.0054%) thiab D (4, 9 mg, 0.0015 feem pua ). Fr. 2-6 (460 mg) tau ntxiv los ntawm HPLC [MeOH–H2O (45 : 55, v/v)] los muab kankanoside E (6, 161 mg, 0.027%) thiab (2E,6Z){192 }}bD-glucopyranosyloxy{195}}, 6-dimethyl-2, 6-octadienoic acid (18, 17 mg, 0.0028%). Tshooj 3 (7.60 g) tau raug thim rov qab-theem silica gel col umn chromatography [230 g, MeOH–H2O (20 : 80 → 40 : 60 → 50 : 50 → 60 : 40, v/v) → MeOH] muab tsib fractions [Fr. 3-1 (2652 mg), Fr. 3-2 (593 mg), Fr. 3-3 (3610 mg), Fr. 3-4 (190 mg), thiab Fr. 3-5 (336 mg)]. Fr. 3-1 (480 mg) raug ntxuav los ntawm HPLC [MeOH–H2O (10 : 90, v/v)] muab kankanoside B (2, 19 mg, 0.018%) , geniposidic acid (8, 32 mg, 0.030 feem pua ​​), thiab ajugol (12, 12 mg, 0.011 feem pua). Tshooj 4 (13.10 g) tau raug rau reversed-phase silica gel kab chromatography [390 g, MeOH–H2O (10 : 90 → 20 : 80 → 30 : 70 → 40 : 60 → 50 : 50, v/v) → MeOH] muab xya feem [Fr. 4-1 (6114 mg), Fr. 4-2 (430 mg), Fr. 4-3 (1058 mg), Fr. 4-4 (170 mg), Fr. 4-5 (2595 mg), Fr. 4-6 (1635 mg), thiab Fr. 4-7 (1064 mg)]. Fr. 4-2 (430 mg) tau muab cais ntxiv los ntawm HPLC [MeOH–H2O (5 : 95, v/v)] kom them taus 2 (70 mg, 0.012%) thiab kankanoside C (3, 16 mg, 0.0027%) . Fr. 4-6 (1058 mg) kuj tau sib cais los ntawm HPLC [MeOH–H2O (15 : 85, v/v)] rau fur nish mussaenosidic acid (7, 116 mg, 0.020%) thiab 8-epiloganic acid (9, 193 mg, 0.033 feem pua). Tshooj 5 (15.15 g) tau raug thim rov qab-theem silica gel kab chromatography [455 g, MeOH–H2O (0 : 100 → 10 : 90 → 20 : 80 → 40 : 60 → 50 : 50, v/v) → MeOH ] muab xya feem [Fr. 5-1 (9311 mg), Fr. 5-2 (1114 mg), Fr. 5-3 (306 mg), Fr. 5-4 (347 mg), Fr. 5-5 (1620 mg), Fr. 5-6 (1453 mg), thiab Fr. 5-7 (1106 mg)]. Fr. 5-1 (9311 mg) tau crystallized los ntawm MeOH kom muab D-mannitol (3337 mg, 4.19 feem pua).

Cov ntsiab lus paub (7-18) tau txheeb xyuas los ntawm kev sib piv ntawm lawv cov ntaub ntawv lub cev ([a]D, IR, 1 H-NMR, 13C-NMR, MS) nrog cov nqi tshaj tawm 1,7,19-24,26,27) lossis cov qauv coj mus muag.25) Kankanoside A (1): Cov hmoov amorphous, [a]D 25 107.4 degree (c 1.50, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C16H26O8Na (M Na) 369.1525; Pom 369.1522. IR (KBr): 3410, 2940, 1647, 1076 cm 1. 1 H-NMR (500 MHz, CD3OD thiab pyridine-d5) d: muab rau hauv Table 1. 13C-NMR (125 MHz, CD3OD thiab pyridine-d5) d C: muab rau hauv Table 2. Positive-ion FAB-MS: m /z 369 (M Na). Negative-ion FAB-MS: m/z 345 (MH).

Kankanoside B (2): Ib qho amorphous hmoov, [a]D 26 118.7 degree (c 0.10, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C15H24O10Na (M Na) 387.1267; Pom 387.1261. IR (KBr): 3410, 2940, 1647, 1085 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab nyob rau hauv Table 1. 13C-NMR (125 MHz, CD3OD) d C: muab nyob rau hauv Table 2. Positive-ion FAB-MS: m/z 387 (M Na). Negative-ion FAB-MS: m/z 363 (MH).

Kankanoside C (3): Ib qho amorphous hmoov, [a]D 26 34.0 degree (c 1.00, MeOH). High-resolution negative-ion FAB-MS: Calcd rau C15H24ClO10 (MH) 399.1058; Pom 399.1077. IR (KBr): 3410, 2964, 1159, 1078, 1048, 949 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab hauv Table 1. 13C NMR (125 MHz, CD3OD) d C: muab hauv Table 2. Negative-ion FAB-MS: m/z 399, 401 (MH).

Kankanoside D (4): Ib qho amorphous hmoov, [a]D 25 30.6 degree (c 0.50, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C15H26O7Na (M Na) 341.1204; Pom 341.1210. IR (KBr): 3410, 2940, 1655, 1078, 1040 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab hauv Table 3. 13C-NMR (125 MHz, CD3OD) d C: muab hauv Table 4. Positive-ion FAB-MS: m/z 341 (M Na). Negative-ion FAB-MS: m/z 317 (MH).

Kankanol (5): Ib qho amorphous hmoov, [a]D 25 11.1 degree (c 1.40, MeOH). High-resolution CI-MS: Calcd rau C9H14ClO4 (MH) 221.0580; Pom 221.0582. IR (KBr): 3399, 3004, 1165, 1096, 1059, 1048, 955 cm 1. 1 H- NMR (500 MHz, CD3OD) d: muab hauv Table 1. 13C-NMR (125 MHz, CD3OD) d C: muab hauv Table 2. CI-MS m/z (%) : 221 (MH) (5 ), 223 (MH) (2), 185 (88), 167 (100), 149 (71), thiab 57 (49).

Kankanoside E (6): Ib qho amorphous hmoov, [a]D 25 20.0 degree (c 2.00, MeOH). High-resolution positive-ion FAB-MS: Calcd rau C16H28O8Na (M Na) 371.1682; Pom 371.1690. UV [MeOH, nm (log e)]: 215 (4.16). IR (KBr): 3410, 2940, 1647, 1085, 1043 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab nyob rau hauv Table 3. 13C-NMR (125 MHz, CD3OD) d C: muab nyob rau hauv Table 4. Positive-ion FAB-MS: m/z 371 (M Na). Negative-ion FAB-MS: m/z 347 (MH).

cistanche extract txiaj ntsig

Acid Hydrolysis ntawm 1-4 thiab 6 nrog 1 M HCl

Kev daws ntawm 1-4 lossis 6 (txhua 1.5 mg) hauv 1 M HCl (0.5 ml) tau ua kom sov hauv qab reflux rau 3 teev. Tom qab txias, cov tshuaj tiv thaiv sib tov tau nchuav rau hauv dej khov thiab nruab nrab nrog Amberlite IRA-400 (OH daim ntawv), thiab cov resin raug tshem tawm los ntawm kev pom. Tom qab ntawd, cov lim tau muab rho tawm nrog EtOAc. Lub aqueous txheej tau raug rau HPLC tsom xam raws li nram no: HPLC kem, Ka sensor LC NH{{10}}, 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 (75: 25, v/v); tus nqi sib tw 0.8 ml / min; kem kub, chav tsev kub. Kev txheeb xyuas ntawm D-glucose tam sim no hauv txheej aqueous tau ua los ntawm kev sib piv ntawm nws lub sijhawm khaws cia thiab kev hloov kho qhov muag nrog rau cov qauv tseeb: tR 12.3 min (zoo kho qhov muag rotation)

Enzymatic Hydrolysis ntawm 1, 4, thiab 6 nrog b-Glucosidase

Kev daws ntawm 1 (7.7 mg) hauv H2O (1.5 ml) tau kho nrog b-glucosidase (5.0 mg, los ntawm almond, Oriental Yeast Co., Tokyo, Nyiv) thiab cov tshuaj tau nplawm ntawm 37 degree. rau 3d. Tom qab EtOH tau ntxiv rau cov tshuaj tiv thaiv sib tov, cov kuab tshuaj tau muab tshem tawm hauv qhov txo qis thiab cov seem raug ntxuav los ntawm HPLC [MeOH–H2O (55: 45, v/v)] los muab kankagenin a (1a, 2.3 mg, 56%). . Los ntawm cov txheej txheem zoo sib xws, (R)-rotundiol33,34) (4a, 1.2 mg, 69%) thiab (2E,6R)-8-hydroxy-2, 6-dimethyl{30} octanoic acid36) (6a, 6.8 mg, 62 feem pua) tau txais los ntawm 4 (3.5 mg) thiab 6 (20.4 mg), feem.

Kankagenin a (1a): Cov hmoov dawb, [a]D 25 18.4 degree (c 0.20, MeOH). High-resolution EI-MS: Calcd rau C10H16O3 (M ) 184.1099; Pom 184.1106. IR (KBr): 3410, 2940, 1684 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab rau hauv Table 1. 13C-NMR (125 MHz, CD3OD) d C: muab hauv Table 2. EI MS: m/z (%) : 184 (M , 37) , 95 (100).

Kev kho Alkaline ntawm 14 nrog 5 feem pua ​​aqueous KOH

Kev daws ntawm 14 (23.0 mg) hauv 5 feem pua ​​aqueous KOH (1.0 ml) tau nplawm ntawm 80 degree rau 2 teev. Cov tshuaj tiv thaiv sib xyaw yog neutralized nrog Amberlite HCR-W2 (H daim ntawv). Kev hloov pauv ntawm cov kuab tshuaj los ntawm cov lim dej hauv qhov txo qis tau muab cov khoom seem, uas tau ua kom huv los ntawm HPLC [MeOH–H2O (5: 95, v/v)] muab 2 (4.0 mg, 16 feem pua) thiab 1932) (10.5 mg. , 43 feem pua).

Acid kho ntawm 14 nrog 5 feem pua ​​aqueous HCl

Kev daws ntawm 14 (25.0 mg) hauv 5 feem pua ​​aqueous HCl (2.0 ml) tau nplawm ntawm chav tsev kub rau 3 teev. Cov tshuaj tiv thaiv sib tov tau nchuav rau hauv dej khov thiab tag nrho cov tshuaj tiv thaiv sib xyaw tau muab rho tawm nrog EtOAc. Lub EtOAc extract tau ua tiav ntxuav nrog dej saturated NaHCO3 thiab brine, tom qab ntawd qhuav dhau los ntawm MgSO4 hmoov thiab lim. Kev tshem tawm cov kuab tshuaj los ntawm cov filtrate nyob rau hauv lub siab txo tau muab ib qho residue, uas tau cais los ntawm HPLC [MeOH–H2O (20: 80, v/v)] muab 14a (4.0 mg, 25 feem pua).

14a

Cov hmoov dawb, [a]D 20 21.5 degree (c 0.30, MeOH). High-resolution CI-MS: Calcd rau C9H14ClO4 (MH) 221.0580; Pom 221.0587. IR (KBr): 3410, 2962, 1365, 1152, 1055, 945 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab hauv Table 1. 13C-NMR (125 MHz, CD3OD) d C: muab hauv Table 2. CI-MS: m/z (%) : 221 (MH) ( 7), 223 (MH) (3), 203 (M H2O) (97), 205 (M H2O) (33), 185 (7), 167 (12), 159 (32), 121 (27), 110 (48), 95 (64), 85 (100), 67 (56), thiab 57 (65).

Acetylation ntawm 5 thiab 14a

Ib qho kev daws ntawm 5 (2.5 mg) hauv pyridine (0.5 ml) tau kho nrog acetic anhydride (Ac2O, 0.4 ml) thiab sib tov sib xyaw ntawm chav tsev kub rau 12 teev. Cov tshuaj tiv thaiv sib tov tau nchuav rau hauv dej khov thiab tag nrho cov tshuaj tiv thaiv sib xyaw tau muab rho tawm nrog EtOAc. EtOAc extract tau ua tiav ntxuav nrog 5 feem pua ​​aqueous HCl, saturated aqueous NaHCO3, thiab brine ces qhuav dhau anhydrous MgSO4 hmoov thiab lim. Kev tshem tawm cov kuab tshuaj los ntawm cov filtrate nyob rau hauv lub siab txo tau muab ib tug residue, uas yog purified los ntawm HPLC [MeOH–H2O (35: 65, v/v)] muab 5a (2.3 mg, 77 feem pua). Los ntawm cov txheej txheem zoo sib xws, 14b (2.7 mg, 87 feem pua) kuj tau npaj thiab ntxuav los ntawm HPLC [MeOH–H2O (55: 45, v/v)] los ntawm 14a (2.1 mg).

5a

Cov hmoov dawb, [a]D 20 1.8 degree (c 0.18, MeOH). High-resolution CI MS: Calcd rau C11H15O5 (MH) 227.0919; Pom 227.0925. IR (KBr): 2962, 1734, 1374, 1258, 1237, 1169, 1103, 1053, 947 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab rau hauv Table 1. 13C-NMR (125 MHz, CD3OD) d C: muab hauv Table 2. CI-MS m/z (%) : 227 (MH) (28 209 (MH2O) (4), 184 (3), 166 (45), 149 (22), 138 (38), 122 (44), 94 (31), 85 (100), thiab 57 (34) ).

14b

Cov hmoov dawb, [a]D 20 23.7 degree (c 0.06, MeOH). High-resolution CI-MS: Calcd rau C13H18ClO6 (MH) 305.0792; pom 305.0790. IR (KBr): 1744, 1376, 1243, 1231, 1001, 941 cm 1. 1 H-NMR (500 MHz, CD3OD) d: muab hauv Table 1. 13C-NMR (125 MHz, CD3OD) d C: muab hauv Table 2. CI-MS: m/z (%) : 305 (MH) ( 2), 307 (MH) (1), 263 (MH C2H3O) (6), 265 (MH C2H3O) (3), 245 (30), 203 (8), 185 (100), 167 (7), 149 (33), 121 (26), 95 (15), 85 (37), thiab 57 (93).

Kev lees paub

Qhov kev tshawb fawb no tau txais kev txhawb nqa los ntawm 21st COE Program, Kev Kawm Frontier Project, thiab Nyiaj Pab rau Kev Tshawb Fawb Txog Kev Tshawb Fawb los ntawm Ministry of Education, Culture, Sports, Science, thiab Technology ntawm Nyiv. Cov kws sau ntawv ua tsaug rau xibfwb Xiaoguang Jia hauv Xinjiang Lub Tsev Kawm Ntawv ntawm Cov Tshuaj Suav thiab Ethnologic Tshuaj hauv Urumqi, Tuam Tshoj, rau kev txheeb xyuas cov khoom cog.

cov txiaj ntsig cistanche

Cov ntaub ntawv thiab Cov Lus Qhia

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