Steroidal Saponins from Fritillaria Pallidiflora Schrenk

Fitoterapia 83 (2012) 785–794

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Fitoterapia

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Steroidal saponins from Fritillaria pallidiﬂora Schrenk

Shuo Shen a, Guoyu Li b, Jian Huang a, Chaojun Chen c, Bu Ren c,GaLuc, Yong Tan b, Jiaxu Zhang a, Xian Li a, Jinhui Wang a,b,c,d,⁎ a School of Traditional Chinese Materia Medica 49#, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China b School of Pharmacy, Shihezi University, Shihezi 832002, PR China c School of Pharmacy, Inner Monglia Medical College, Hohhot, 010110, PR China d Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016, PR China article info abstract

Article history: Five new steroidal saponins, Pallidifloside D (1), Pallidifloside E (2), Pallidifloside G (5), Palli- Received 28 November 2011 difloside H (6) and Pallidifloside I (7), together with seven other steroidal saponins (3, 4, 8–12) Received in revised form 2 March 2012 were isolated from the dry bulbs of Fritillaria pallidiflora Schrenk. Their structures were estab- Accepted 7 March 2012 lished by spectroscopic techniques (IR, MS, 1D and 2D NMR) and chemical means. The isolated Available online 16 March 2012 steroidal saponins were evaluated for cyotoxic activity against human C6 brain gliomas and Hela cervix cancer cell lines using MTT assays. Compounds 1, 10, 11, 12 showed cytotoxicity Keywords: against C6 and Hela cell lines with IC50 values in the range of 5.1–75.8 μM. Fritillaria pallidiflora Schrenk © 2012 Elsevier B.V. All rights reserved. Steroidal saponins NMR Cytotoxic activity

1. Introduction pallidiflora had been investigated. Five new steroidal saponins, pallidiflosides D (1), E (2), G (5), H (6) and I (7) (see Fritillaria pallidiflora Schrenk belongs to the Fritillaria Fig. 1), together with seven other steroidal saponins namely genus of Liliaceae family widely distributed in Xinjiang prov- Spongipregnoloside A (3) [7], Smilaxchinoside C (4) [5], ince of China and finds widespread applications as antitus- Timosaponin H1 (8) [8], Protobioside (9) [9], Polygonatoside sive, antiasthmatic and expectorant medicine [1]. The B3 (10) [10], Polyphyllin V (11) [11] and Deltonin (12) [12] chemical constituents of F. pallidiflora have been studied, were isolated from the dry bulbs of F. pallidiflora Schrenk. and steroidal saponins and alkaloids are regarded as their This paper deals with their structural elucidation and cyto- main ingredients [2]. In recent years, steroidal saponins toxic activity against human C6 brain gliomas and Hela cervix have attracted more attention for their significant bioactiv- cancer cell lines using MTT assays. ities, including their anti-tumor [3], anti-thrombotic [4], anti-inflammatory [5], anti-fungal [6] activities. However, a 2. Experimental part literature survey concerning the secondary metabolites of F. pallidiflora showed that no systematic chemical work on the 2.1. General experimental procedures saponin constituents had been carried out on the plant. In order to screen the bioactive steroidal saponins for develop- Optical rotations were determined on a Perkin-Elmer 241 ing new medicinal resources, the chemical constituents of F. MC polarimeter. IR spectra were recorded on a Shimadzu IRPrestige-21 spectrophotometer. The NMR data were recorded on a Varian INOVA-300 spectrophotometer (300 MHz for 1H ⁎ Corresponding author at: School of Traditional Chinese Materia Medica and 75 MHz for 13C) in C D N. The ESI-MS data were obtained 49#, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 5 5 110016, PR China. Tel./fax: +86 024 23986479. on a Finnigan LCQ mass spectrometer. The HR-ESI-MS data E-mail address: [email protected] (J. Wang). were obtained on a Waters LCT Premier XE time-of-flying

21 21 20 O O 20 18 12 18 22 17 H 12 17 11 16 O 19 13 11 H 19 13 16 1 9 H H 15 1 9 2 14 15 10 8 2 14 H H 10 8 3 H H 7 3 R1O 5 7 4 6 R1O 5 4 6 2R1=S1 1R1=S2 3R1=S2

R2O R2O 21 OH 23 26 25 23 26 25 20 22 21 20 O O 18 24 27 H 18 22 24 27 12 17 H O 12 17 11 O 19 13 16 11 H H 19 13 16 1 9 H H 1 9 15 2 14 15 10 8 2 14 H H 10 3 H 8 H 7 3 R1O 5 7 4 6 R1O 5 4 6

4R1=S2,R2=S4 5R1=S1,R2=S4 6R1=S1,R2=S4

R2O 25 O 26 21 HO 21 23 26 25 27 20 20 23 24 18 22 18 22 H 24 27 H 12 17 12 17 O O 11 11 19 13 16 19 13 16 H H H R2 1 9 1 9 2 14 15 2 14 15 8 10 8 10 H H H H 3 3 7 5 7 R1O 5 R1O 4 6 4 6 10 R =S ,R=OH 7R1=S1,R2=S4 1 2 2 8R1=S5,R2=S4 11 R1=S2,R2=H 9R1=S2,R2=S4 12 R1=S3,R2=H

OH OH O O O HO O HO HO HO HO OH 1''' O 1' = S = O 1' S1 2 O 1'' HO HO O 1''

HO HO OH OH OH OH O O HO O OH HO HO S = O S = OH 1''' O 1' 4 3 HO HO 1'''' HO O 1'' OH

HO OH OH O O O HO O OH HO HO S5= OH 1'''OH O 1'' O O HO HO OH 1' HO 1'''' OH

Fig. 1. Structures of compounds 1–12. S. Shen et al. / Fitoterapia 83 (2012) 785–794 787

mass spectrometer. HPLC (Shimadzu-LC-8A pump, Shimadzu- MeOH–H2O (65:35) as mobile phase to afford compound 3 SPD-20A UV spectrophotometric detector, Shimadzu Shim- (34.1 mg, , tR=28.0 min) and 12 (18.3 mg, tR=43.2 min). pack PRC-ODS 10 μm, 20×250 mm preparation column) was Fraction 8 (8.5 g) was charomatographed on RP-18 column used for separation. Column chromatography was performed chromatography using as eluents MeOH–H20mixtures(1:9; on silica gel (200–300 mesh; Qingdao Marine Chemical 3:7; 5:5; 6:4; 9:1, each 500 ml) and finally MeOH (700 ml). Group, Co., Qingdao, China) and ODS (40–63 μm, Merck). GC After evaporation of the solvents, six fractions were obtained:

(gas chromatography) analysis was performed on a FULI GC- Fr8-1 (MeOH–H20, 1:4) (2.3 g), Fr8-2 (MeOH–H20, 2:5) (1.6 g), 9790 gas chromatograph equipped with an H2 flame ionization Fr8-3 (MeOH–H20, 5:5) (1.3 g), Fr8-4 (MeOH–H20, 6:4) (1.8 g), detector and a SE-30 quartz capillary column (30 m×0.25 Fr8-5 (MeOH–H20, 4:1) (0.8 g), Fr8-6 (MeOH) (0.3 g). Fr8-3 mm×0.25 μm). Absorbance value was measured by a Bio-Rad (1.3 g) was further purified by preparative HPLC (column: Model 680 (Bio-Rad Laboratories Inc., CA, USA) to calculate 20×250 mm, RP-18, 10 μm, flow rate: 12.0 ml/min, 210 nm) the inhibition rate. with MeOH–H2O (57:43) as mobile phase to afford compound 1 (8.0 mg, tR=24.7 min), 2 (12.1 mg, tR=43.7 min),5(10.1 mg, t =15.3 min) and 6 (29.4 mg, t =11.2 min). Fr8-4 (1.8 g) 2.2. Plant material R R was further purified by preparative HPLC (column: 20×250 mm, RP-18, 10 μm, flow rate: 12.0 ml/min, 210 nm) with The dry bulbs of F. pallidiflora Schrenk (2.0 kg) were col- MeOH–H O (65:35) as mobile phase to afford compound 7 lected in Yining, Xinjiang province of China in June 2008, 2 (45.1 mg, t =20.3 min), 8 (9.6 mg, t =17.4 min), 9 (26.3 mg, and identified by Dr Yong Tan of Shihezi University. A vouch- R R t =19.4 min). er specimen (No.2008090158) is deposited in Research De- R partment of Natural Medicine, Shenyang Pharmaceutical 2.4. Cytotoxicity assay University. Human C6 brain gliomas and Hela cervix cancer cell lines

2.3. Extraction and Isolation were cultivated in humidified incubator (5% CO2 and 37 °C) and these cells were grown in DMEM (Dulbecco's Modified The dry bulbs of F. pallidiflora Schrenk (2.0 kg) were Eagle Medium) medium containing 10% FBS (Fetal Bovine extracted with 70% EtOH (3×20 L) for 3 h at 95 °C. The extracts Serum) with L-glutamine. The human C6 and Hela cancer cells were combined and concentrated under reduced pressure to in the log phase of their growth cycle (5×104 cells/ml) were give the brown residue (98.0 g). The residue was suspended in added to each well of the 96-well plates (100 μL/well) were cul- water (2 L) and then extracted with petroleum ether (2 L), tivated for 12 h. The cells were then treated with compounds of

CHCl3 (2 L), EtOAc (2 L) and n-BuOH (2 L), respectively. The n- six concentrations (3, 6.25, 12.5, 25, 50, and 100 μM) and incu- BuOH layer were concentrated under reduced pressure to give bated in a humidified atmosphere of 5% CO2 for 48 h at 37 °C. the brown extract (26.0 g). The n-BuOH extract (26.0 g) was After the cells culture had been terminated, 10 μL MTT (3- chromatographed on silica gel eluting with gradient elution by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

CHCl3/MeOH (100:1–100:100) to yield ten fractions, 1–10. 5 mg/ml) was added to each well, and the cells were incubated Fraction 5 (2.6 g) was charomatographed on RP-18 column for additional 4 h in a humidified atmosphere of 5% CO2 at 37 °C. chromatography using as eluents MeOH–H20 mixtures (1:9, Thereafter, the supernatant was discarded and after washing 3:7, 5:5, 7:3, 9:1, each 500 ml) and finally MeOH (500 ml). with PBS, DMSO (100 μL per well) was added to dissolve the After evaporation of the solvents, six fractions were obtained: formazan crystals. The plate was mixed on a microshaker for

Fr5-1 (MeOH–H20, 1:9) (0.2 g), Fr5-2 (MeOH–H20, 3:7) 15 min and then the absorbance was measured at 490 nm by (0.1 g), Fr5-3 (MeOH–H20, 5:5) (0.3 g), Fr5-4 (MeOH–H20, enzyme immunoassay instrument (Bio-Rad Model 680). 7:3) (1.2 g), Fr5-5 (MeOH–H20, 9:1) (0.2 g), Fr5-6 (MeOH) (0.2 g). Fr5-4 (1.2 g) was further purified by reversed-phase 2.5. Acid hydrolysis of 1, 2, 5, 6 and 7 preparative HPLC (column: 20×250 mm, RP-18, 10 μm, flow rate: 12.0 ml/min, 210 nm) with MeOH–H2O (75:25) as mobile Each compound (2.0 mg) was hydrolyzed with 2 M HCl phase to afford compound 10 (14.1 mg, tR =65.0min). Fr5-5 (2.0 ml), heated for 2 h at 95.2 °C and extracted with CHCl3 (MeOH–H20, 9:1) (0.2 g) was further purified by reversed- (3×5.0 ml). The aqueous layer was concentrated to dryness phase preparative HPLC (column: 20×250 mm, RP-18, 10 μm, under a stream of nitrogen. The aqueous residue was redis- flow rate: 12.0 ml/min, 210 nm) with MeOH–H2O (86:24) as solved in anhydrous pyridine (1 ml) and L-cysteine methyl mobile phase to afford compound 11 (9.2 mg, tR =107.1 min). ester hydrochloride (2.0 mg) was added to the solution of pyri- Fraction 6 (1.7 g) was charomatographed on RP-18 column dine. The mixture was heated at 60 °C for 2 h and 0.5 ml TMSI chromatography using as eluents MeOH–H20mixtures(1:9, (N-Tremethylsilyimidazole) was added, followed by heated at 3:7, 5:5, 6:4, 8:1, each 500 ml) and finally MeOH (500 ml). 60 °C for 2 h. The reaction product was analyzed on a GC analy- After evaporation of the solvents, six fractions were obtained. sis under the following conditions: GC: FULI GC-9790 equipped

Fr6-3 (MeOH–H20, 5:5) (0.4 g) was further purified by with an H2 flame ionization detector (FID) and a SE-30 quartz reversed-phase HPLC (column: YMC-pack ODS, 6×150 mm RP- capillary column (30 m×0.25 mm×0.25 μm), Column temper-

18, 5 μm, flow rate: 2.0 ml/min, 210 nm) with MeOH–H2O ature: 120–280 °C with the rate of 10 °C/min, carrier gas: N2 (58:42) as mobile phase to afford compound 4 (8.5 mg, (30 ml/min), split ratio: 30:1, injection temperature: 280 °C, de- tR=19.5 min). Fr6-4 (MeOH–H20, 6:4) (0.7 g) was further puri- tector temperature: 290 °C, injection volume: 1.0 μL. The mono- fied by reversed-phase preparative HPLC (column: 20×250 saccharides of compound were confirmed by comparison of the mm, RP-18, 10 μm, flow rate: 12.0 ml/min, 210 nm) with retention time of monosaccharides derivatives with those of 788 S. Shen et al. / Fitoterapia 83 (2012) 785–794

−1 standard sugars (the standard sugars were subjected to the 0.51); IR banks (KBr) νmax (cm ): 3416, 2935, 2907, 1733, same reaction). 1710, 1638, 1380, 1363, 1073, 1045, 951, 912, 832; HR-ESI-MS − + m/z: 1049.5166 [M–H] (calculated for C50H81O23 1049.5169). 2.6. Data of compounds ESI-MS m/z: 1071 [M+Na]+,939[(M+Na+)−132]+, 925 [(M+Na+)−146]+,909[(M+Na+)−162]+, 631 [(M+ 2.6.1. Pallidifloside D Na+)−132−146−162]+.1HNMRand13CNMRdatasee 20 (1): white amorphous solid; [α]D =−50.9 (MeOH, c Table 4. − 1 0.51); IR banks (KBr) νmax (cm ): 3426, 2935, 1752, 1650, 1387, 1286, 1200, 1049, 1040, 914, 882, 844; HR-ESI-MS m/ 2.6.5. Pallidifloside I + + z: 651.3389 [M+H] (calculated for C34H51O12 651.3381). (7): white amorphous solid; positive Liebermann–Burchard + + + 1 20 ESI-MS m/z: 673 [M+Na] , 527 [(M+Na )−146] . H and positive Ehrlich reagent tests; [α]D =−54.9 (MeOH, c 13 −1 NMR and C NMR data see Table 1. 0.52); IR banks (KBr) νmax (cm ): 3423, 2948, 2908, 1654, 1379, 1161, 1072, 1043, 894, 838; HR-ESI-MS m/z: 1035.5379 + + 2.6.2. Pallidifloside E [M+H] (calculated for C50H83O22 1035.5376). ESI-MS m/z: 20 + + + + (2): white amorphous solid; [α]D =−68.5 (MeOH, c 1057[M+Na] , 925[(M+Na )−132] , 911[(M+Na )− −1 + + + + 0.50); IR banks (KBr) νmax (cm ): 3405, 2936, 1654, 1374, ek146] , 895[(M+Na )−162] , 617[(M+Na )−132− 1364, 1204, 1071, 1039, 949, 909, 889, 840; HR-ESI-MS m/z: 146−162]+.1HNMRand13CNMRdataseeTable 5. − − 753.3699 [M–H] (calculated for C38H57O15 753.3697). ESI- MS m/z:777[M+Na]+, 645 [(M+Na+)−132]+, 631 [(M+ 3. Results and discussion Na+)−146]+, 337 [(M+Na)+−132−146−162]+. 1H NMR and 13C NMRdata see Table 2. Compound 1 was obtained as white amorphous solid 20 with [α]D −50.9 (MeOH, c 0.51). The IR spectrum showed 2.6.3. Pallidifloside G absorptions for carbonyl ester (1752 cm− 1), hydroxyl (5): white amorphous solid; positive Liebermann–Burchard (3426 cm− 1) and the glycosidic linkage (1000–1150 cm− 1) 20 and positive Ehrlich reagent tests; [α]D =−66.8 (MeOH, c [18]. Its molecular formula, C34H50O12 was deduced from −1 + 0.51); IR banks (KBr) νmax (cm ): 3420, 2936, 2906, 1652, the HR-ESI-MS: (m/z 651.3389, [M+H] ). The ESI-MS spec- 1638, 1366, 1074, 1039, 913, 838; HR-ESI-MS m/z: 1033.5212 trum in positive-ion mode showed m/z 673 [M+Na]+, 527 + + + + 1 [M+H] (calculated for C50H81O22 1033.5219). ESI-MS m/z: [(M+Na )−146] . The H NMR spectrum of 1 showed 1055 [M+Na]+,923[(M+Na+)−132]+, 909 [(M+Na+)− two methyl singlet signals at δ 0.60 (s) and 1.02 (s), one 146]+,893[(M+Na+)−162]+,615[(M+Na+)−132−146− methyl doublet signal at δ 1.81 (d, J=6.3 Hz), three olefinic 162]+. 1H NMR and 13C NMR data see Table 3. proton signals at δ 5.29 (d, J=4.1), 6.38 (s) and 5.54 (s), and two anomeric pronton signals at δ 5.08 (d, J=7.8 Hz) 2.6.4. Pallidifloside H and 6.46 (br s). These data, combined with the analysis of (6): white amorphous solid, positive Liebermann–Burchard its 13C NMR spectrum (two angular methyl group at δ 14.0 20 and positive Ehrlich reagent tests; [α]D =−56.2 (MeOH, c and 19.1, a trisubstituted double bonds at δ 140.5 and

Table 1 1 13 1 13 a The H and C NMR data for compounds 1 (Pyridine-d5, H NMR 300 MHz, C NMR 75 MHz, δ in ppm, J in Hz) .

No. 1 No. 1

δC δH δC δH

1 37.1 0.91 m, 1.68 (br d, 10.3) (3-O)-β-D-Glc 100.0 5.08 (d, 7.8) 2 29.8 1.87 m, 2.13 m 2′ 79.4 4.33 (dd, 8.9, 7.8) 3 77.5 3.92 m 3′ 78.1 4.32 m 4 38.6 2.82 (br d, 13.2), 2.75 m 4′ 71.5 4.27 (t, 9.0) 5 140.5 5′ 77.5 3.95 m 6 121.1 5.29 (d, 4.1) 6′ 62.3 4.38 (m), 4.56 (br d, 11.2) 2 7 31.8 1.87 m, 1.46 m (Glc )-α-L-Rha 101.8 6.46 br s 8 31.2 1.44 m 2″ 72.3 4.86 (br d, 3.0) 9 49.8 0.92 m 3″ 72.6 4.69 (dd, 9.3, 3.0) 10 36.8 4″ 73.9 4.42 (t, 9.3) 11 20.3 1.26 m, 1.43 m 5″ 69.3 5.03 (m) 12 37.7 1.07 m, 1.67 (br d, 12.6) 6″ 18.4 1.81 (d, 6.3) 13 43.5 14 54.3 1.02 m 15 33.0 2.17 m, 1.52 (overlap) 16 81.5 4.86 m 17 54.8 2.74 (d, 8.4) 18 14.0 0.60 s 19 19.1 1.02 s 20 137.3 21 121.6 6.38 s, 5.54 s 22 171.0 a The assignments were based on 1H–1H COSY, HMQC and HMBC experiments. S. Shen et al. / Fitoterapia 83 (2012) 785–794 789

Table 2 1 13 1 13 a The H and C NMR data for compounds 2 (Pyridine-d5, H NMR 300 MHz, C NMR 75 MHz, δ in ppm, J in Hz) .

No. 2 No. 2

δC δH δC δH

1 37.4 0.90 m, 1.69 m (3-O)-β-D-Glc 100.0 4.99 (d, 7.1) 2 30.2 1.85 m, 2.15 (br d, 12.8) 2′ 77.6 4.23 m 3 78.1 3.88 m 3′ 77.3 4.25 m 4 39.0 2.78 m 4′ 81.5 4.20 m 5 141.2 5′ 76.3 3.85 m 6 121.6 5.33 (d, 4.8) 6′ 61.6 4.36 (dd, 11.4, 5.1), 4.54 m 2 7 31.8 1.90 m (Glc )-α-L-Rha 102.0 6.34 (br s) 8 30.3 1.51 m 2″ 72.5 4.83 (br d, 3.3 ) 9 50.8 0.92 m 3″ 72.9 4.65 (dd, 9.3, 3.0) 10 37.2 4″ 74.2 4.42 (t, 9.3,) 11 20.9 1.46 m 5″ 69.6 4.95 m 12 35.1 1.40 m, 2.65 (dt 11.7, 3.4) 6″ 18.7 1.80 (d, 6.3) 4 13 46.3 (Glc )-β-D-Xyl 105.8 5.06 (d, 7.5) 14 56.4 1.43 m 2″ 75.0 4.00 (dd, 8.9,7.5) 15 32.3 1.96 m, 2.21 (ddd, 13.8, 6.6, 3.2) 3″ 78.4 4.00 m 16 144.8 6.61 (dd, 3.2, 1.7) 4″ 70.8 4.13 m 17 155.2 5″ 67.4 3.71 (t, 10.8), 4.31 m 18 15.9 0.95 s 19 18.7 1.07 s 20 196.3 21 27.1 2.26 s a The assignments were based on 1H–1H COSY, HMQC and HMBC experiments.

121.1, an exocyclic double bonds at δ 121.6 and 137.3, an dihydroxypregn-5-ene-20-carboxylic acid γ-lactone. In the ester C=O signal at δ 171.0, and two anomeric carbon signals NMR spectra of 1, the signals of a methyl group (C-21) and at δ 100.0 and 101.8) indicated the aglycone of 1 was a C-22 methenyl group (C-20) were not observed, but the signals steroidal lactone. A comparison of the NMR spectral data of 1 of an exocyclic double bonds at δ 121.6 and 137.3 (C-20 and (Table 1) with literature data [13] indicated the aglycone of 1 C-21) were observed. These were confirmed by HMBC spec- possessed a similar skeleton as (3β,16β, 20S)-3, 16- trum. In the HMBC spectrum, the correlations between the

Table 3 The 1H and 13C NMR data for compounds 5 (Pyridine-d5, 1H NMR 300 MHz, 13C NMR 75 MHz, δ in ppm, J in Hz) a.

No. 5 No. 5

δC δH δC δH

1 37.1 0.92 m, 1.71 m (3-O)-β-D-Glc 99.9 4.98 (d, 7.8) 2 29.9 1.82 m, 2.06 m 2′ 77.2 4.23 m 3 77.8 3.88 m 3′ 77.8 4.24 m 4 38.7 2.78 (dd, 12.8, 3.9), 2.74 (br t, 12.6) 4′ 81.1 4.17 m 5 140.4 5′ 75.6 3.86 m 6 121.4 5.28 (d, 4.0) 6′ 61.3 4.38 m, 4.46 (br d, 12.1) 2 7 31.7 1.46 m, 1.88 m (Glc )-α-L-Rha 101.8 6.33 s 8 30.8 1.53 m 2″ 72.3 4.72 (br d, 3.2) 9 49.8 0.86 m 3″ 72.6 4.63 m 10 36.7 4″ 73.9 4.44 (t, 9.5) 11 20.3 1.41 m 5″ 69.3 4.95 m 12 39.0 1.17 m, 1.86 (overlap) 6″ 18.5 1.81 (d, 6.0) 4 13 40.1 (Glc )-β-D-Xyl 105.5 5.08 (d, 7.5) 14 56.6 0.98 m 2‴ 74.7 4.02 (dd, 8.9, 7.5) 15 33.3 1.52 m, 2.06 m 3‴ 78.1 4.14 m 16 84.0 5.23 (td, 6.6, 3.7) 4‴ 70.5 4.17 m 17 67.5 2.24 (d, 6.5) 5‴ 67.5 3.69 (d, 9.9), 4.28 m 18 13.2 0.92 s (26-O)-β-D-Glc 104.6 4.86 (d, 7.8) 19 19.1 1.09 s 2″″ 75.0 4.03 (dd, 9.0, 7.8) 20 76.4 3″″ 78.4 4.26 m 21 21.6 1.76 s 4″″ 71.3 4.25 m 22 163.4 5″″ 78.3 3.97 m 23 91.4 4.51 (d, 7.0) 6″″ 62.5 4.40 m, 4.53 m 24 29.6 2.27 m, 2.41 m 25 34.7 2.08 m 26 75.0 3.68 m, 4.02 m 27 17.4 1.06 (d, 7.8) a The assignments were based on 1H–1H COSY, HMQC and HMBC experiments. 790 S. Shen et al. / Fitoterapia 83 (2012) 785–794

Table 4 1 13 1 13 a The H and C NMR data for compounds 6 (Pyridine-d5, H NMR 300 MHz, C NMR 75 MHz, δ in ppm, J in Hz) .

No. 6 No. 6

δC δH δC δH

1 37.1 0.94 m, 1.71 (br d, 13.2) (3-O)-β-D-Glc 99.7 5.02 (d, 7.8) 2 29.9 1.88 m, 2.10 m 2′ 77.2 4.23 (dd, 9.0, 7.8) 3 77.8 3.92 m 3′ 77.0 4.24 m 4 38.7 2.78 (dd, 13.2, 4.0), 2.74 m 4′ 81.1 4.26 m 5 140.6 5′ 76.0 3.90 m 6 121.3 5.30 (d, 4.2) 6′ 61.3 4.39 (dd, 11.4, 5.1), 4.48 m 2 7 31.6 1.90 m (Glc )-α-L-Rha 101.7 6.34 s 8 30.6 1.51 m 2″ 72.2 4.83 (br d, 3.3) 9 50.1 0.87 m 3″ 72.5 4.67 (dd, 9.5, 3.3) 10 36.7 4″ 73.9 4.39 m 11 20.3 1.47 m 5″ 69.3 4.98 m 12 37.8 1.07 m, 2.14 (overlap) 6″ 18.4 1.82 (d, 6.3) 4 13 42.0 (Glc )-β-D-Xyl 105.5 5.09 (d, 7.8) 14 53.7 0.84 m 2‴ 74.9 4.03 m 15 35.2 1.34 (td, 13.4, 4.2), 2.48 m 3‴ 78.1 4.14 m 16 74.4 5.69 (td, 7.5, 4.2) 4‴ 70.5 4.16 m 17 66.3 2.50 (d, 7.5) 5‴ 67.1 3.72 (t, 10.0), 4.29 m 18 13.5 1.24 s (26-O)-β-D-Glc 104.7 4.84 (d, 7.8) 19 19.1 1.07 s 2″″ 74.9 4.05 m 20 205.3 3″″ 78.3 4.27 m 21 30.1 2.15 s 4″″ 71.3 4.23 m 22 173.0 5″″ 78.3 3.95 m 23 31.9 2.43 m 6″″ 62.5 4.31 m, 4.52 m 24 28.7 1.56 m, 1.92 m 25 33.1 1.90 m 26 74.7 3.52 m, 3.95 m 27 16.6 0.92 (d, 6.6) a The assignments were based on 1H–1H COSY, HMQC and HMBC experiments.

Table 5 1 13 1 13 The H and C NMR data for compounds 7 (Pyridine-d5, H NMR 300 MHz, C NMR 75 MHz, δ in ppm, J in Hz).

No. 7 No. 7

δC δH δC δH

1 37.2 0.98 m, 1.73 (br d, 12.1) (3-O)-β-D-Glc 99.7 4.97 (d, 7.8) 2 29.9 1.88 m, 2.12 m 2′ 77.2 4.23 (dd, 7.8, 9.0) 3 77.8 3.88 m 3′ 77.0 4.24 m 4 38.7 2.77 (dd, 13.6, 4.0), 2.74 m 4′ 80.8 4.27 m 5 140.4 5′ 76.0 3.92 m 6 121.6 5.28 (d, 3.6) 6′ 61.3 4.40 (dd, 11.2, 5.1), 4.48 m 2 7 32.1 1.47 m, 1.81 m (Glc )-α-L-Rha 101.7 6.31 s 8 31.4 1.51 m 2″ 72.2 4.83 (br d, 3.2) 9 50.0 0.90 m 3″ 72.5 4.62 (dd, 9.5, 3.2) 10 36.9 4″ 73.9 4.40 (t, 9.5) 11 20.8 1.43 m 5″ 69.3 4.96 m 12 39.7 1.08 m, 1.75 (overlap) 6″ 18.5 1.80 (d, 6.3) 4 13 40.4 (Glc )-β-D-Xyl 105.5 5.07 (d, 7.8) 14 56.3 1.06 m 2‴ 74.7 4.03 m 15 32.2 1.47 m, 2.02 m 3‴ 78.1 4.14 m 16 81.1 4.99 m 4‴ 70.5 4.17 m 17 63.6 1.92 (d, 9.5) 5‴ 67.1 3.70 (t, 10.2), 4.29 m 18 16.2 0.93 s (26-O)-β-D-Glc 104.7 4.84 (d, 7.5) 19 19.1 1.06 s 2″″ 74.9 4.05 (dd, 9.0, 7.5) 20 40.5 2.23 (qd, 6.6, 6.4) 3″″ 78.3 4.29 m 21 16.2 1.35 (d, 6.6) 4″″ 71.4 4.23 m 22 110.4 5″″ 78.3 3.95 m 23 37.0 2.03 m 6″″ 62.5 4.31 m, 4.57 m 24 28.1 1.67 m, 2.01 m 25 34.0 1.97 m 26 75.0 3.60 (dd, 9.4, 5.1), 3.93 m 27 17.2 0.99 (d, 6.6) a The assignments were based on 1H–1H COSY, HMQC and HMBC experiments. S. Shen et al. / Fitoterapia 83 (2012) 785–794 791 proton signal at δ 6.38 (s, C-21) and the carbon signals at δ showed three methyl singlet signals at δ 0.92, 1.09 and 1.76; 54.8 (C-17), 137.3 (C-20), 171.0 (C-22) were observed. two methyl doublet signals at δ 1.06 (d, J=7.8 Hz) and 1.81 Thus the aglycone of 1 was identified as (3β,16β, 20S)-3,16- (d, J=6.0 Hz); one olefinic proton at δ 5.28 (d, J=4.0 Hz); dihydroxypregn-5,20-diene-20-carboxylic acid γ-lactone. Acid four anomeric pronton signals at δ 4.98 (d, J=7.8 Hz), 6.33 hydrolysis of 1 afforded rhamnose and glucose. Combined with (s), 5.08 (d, J=7.5 Hz) and 4.86 (d, J=7.8 Hz). These data, its 1Hand13C NMR data, the configurations of the anomeric car- combined with the analysis of its 13C NMR spectrum (three an- bons of sugars were determined to be β-forglucoseandα-L for gular methyl group at δ 13.2, 19.1 and 21.6, two secondary rhamnose [14,15]. The sugar sequences and its linkage to C-3 of methyl groups at δ 17.4, 18.5, two trisubstituted double the aglycone were ascertained by HMBC correlations (Fig. 2)be- bonds at δ 140.4, 121.4 and 163.4, 91.4, and four anomeric car- tween the anomeric proton signal H-1′ at δ 5.08 and the carbon bon signals at δ 99.9, 101.8, 105.5 and 104.6 (Table 3)indicated signal C-3 at δ 77.5, between H-1″ at δ 6.46 and C-2′ at δ 79.4. that 5 was a furostanol saponin with four sugar units. A com- According to the accumulated evidenceabove,thestructureof parison of the NMR spectral data of 5 (Table 3) with literature the 1 was identified as (3β,16β)-3,16-dihydroxypregna-5,20- data [5] allowed the identification of the aglycone as the previ- diene-20-carboxylic acid γ-lactone 3-O-α-L-rhamnopyrano- ously reported 3β,20α,26-trihydroxydurost-5,22-diene. The syl(1→2)-β-D-glucopyranoside, named as pallidifloside D (1). HMBC spectrum (Fig. 2)of5 showed correlations between Compound 2 was obtained as white amorphous solid with the proton signals at δ 4.51 (H-23) and the carbon signals at δ 20 [α]D −68.5 (MeOH, c 0.50). The IR spectrum showed charac- 76.4 (C-20) and 163.4 (C-22); between the proton signals at δ teristic absorptions for carbonyl ester (1654 cm -1), hydroxyl 1.09 (H-19) and the carbon signals at δ 121.4 (C-5), 49.8 (C- (3405 cm−1) and the glycosidic linkage (1000–1150 cm−1) 9) and 36.7 (C-10). The above evidence convinced the presence

[18]. Its molecular formula, C38H58O15 was deduced from the of two double bonds at C-5 (6) and C-22 (23). The geminal pro- − HR-ESI-MS: (m/z 753.3699, [M–H] ). The ESI-MS spectrum ton resonances of H2-26 appeared at δ 3.68 (Ha-26) and 4.02 + in positive-ion mode showed m/z 777 [M+Na] ,645[(M+ (Hb-26), which showed the Δab of δHa-δHb 0.34 ppm Na+)−132]+, 631 [(M+Na+)−146]+, 337 [(M+Na)+ − (b0.48 ppm), indicated the configuration of C-25 was R [16]. 132−146−162]+.The1H NMR spectrum of 2 showed three Acid hydrolysis of 5 afforded rhamnose, glucose and xylose. methyl singlet signals at δ 0.95 (s), 1.07 (s) and 2.26 (s), one Combined with its 1Hand13C NMR data, the configurations methyl doublet signal at δ 1.80 (d, J=6.3 Hz), two olefinic pro- of the anomeric carbons of sugars were determined to be β-D ton signals at δ 5.33 (d, J=4.8) and 6.61 (dd, 3.2, 1.7), and three for glucose, xylose and α-L for rhamnose [14,15].Thesugarse- anomeric pronton signals at δ 4.99 (d, J=7.1 Hz), 6.34 (br s) quences and its linkage to C-3 and C-26 of the aglycone were and 5.06 (d, J=7.5 Hz). The 13C NMR and dept spectra revealed ascertained by HMBC correlations (see Fig. 2). According to 38 carbon signals, of which 17 were assigned to the sugar moi- the accumulated evidence above, the structure of the 5 eties and 21 to the aglycone moiety. One carbonyl carbon at δ was identified as (25R)-26-O-β-D-glucopyranosyl-3β,20α,26- 196.3, two olefinic carbons at δ155.2 and 144.8 indicating the trihydroxydurostan-5,22-diene 3-O-β-D-xylopyranosyl(1→4)- presence of an α, β-unsaturated ketone in the aglycone of 2 [α-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside, named as [7]. Three angular methyl group at δ 15.9, 18.7 and 27.1, one pallidifloside G (5). secondary methyl groups at δ 18.7, one trisubstituted double Compound 6 wasobtainedaswhiteamorphoussolidwith 20 bonds at δ 141.2, 121.6, and three anomeric carbon signals at [α]D −56.2 (MeOH, c 0.51) and it gave positive Liebermann– δ 100.0, 102.0 and 105.8 were also confirmed in the 13CNMR Burchard and positive Ehrlich reagent tests. The IR spectrum and dept spectra. These data indicated that 2 was a pregnane showed characteristic absorptions for hydroxyl (3416 cm−1), glycoside with three sugar units. A Comparison of the NMR carbonyl ester (1733 cm−1) and the glycosidic linkage (1000– −1 spectral data of 2 (Table 2) with literature data [7] indicated 1150 cm ) [18]. Its molecular formula, C50H80O23 was deduced the aglycone pair of 2 was identical with that of pregna-5,16- from the HR-ESI-MS (m/z 1049.5166, [M+H]+). The ESI-MS dien-3β-ol-20-one. Acid hydrolysis of 2 afforded rhamnose, spectrum in positive-ion mode showed m/z 1071 [M+Na]+, glucose and xylose. Combined with its 1Hand13CNMRdata, 939 [(M+Na+)−132]+,925[(M+Na+)−146]+, 909 [(M+ the configurations of the anomeric carbons of sugars were de- Na+)−162]+,631[(M+Na+)−132−146−162]+.The1H termined to be β-D for glucose, xylose and α-L for rhamnose NMR spectrum of 6 showed three methyl singlet signals at δ [14,15]. The sugar sequences and its linkage to C-3 of the agly- 1.24, 1.07 and 2.15; two methyl doublet signal at δ 0.92 (d, cone were ascertained by HMBC correlations (see Fig. 2). J=6.6Hz) and δ 1.82 (d, J=6.3 Hz); one olefinic proton at δ According to the accumulated evidence above, the structure 5.30 (d, J=4.2 Hz); four anomeric pronton signals at δ 5.02 (d, of the 2 was identified as pregna-5,16-dien-3β-ol-20-one 3- J=7.8 Hz), 6.34 (s), 5.09 (d, J=7.8 Hz) and 4.84 (d, J=7.8Hz). 13 O-[β-D-xylopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→4)]- The C NMR spectrum revealed 50 carbon signals, of which 23 β-D-glucopyranoside, named as pallidifloside E (2). were assigned to the sugar moieties and 27 to the aglycone moi- Compound 5 was obtained as white amorphous solid with ety. The above evidences indicated 6 was a furostanol saponin 20 [α]D −66.8 (MeOH, c 0.51) and it gave positive Liebermann– with four sugar units. Comparison of the NMR spectral data of Burchard and positive Ehrlich reagent tests. The IR spectrum 6 (Table 4) with literature data [17] allowed the identification showed absorption bands at 3420, 1074 cm− 1, suggestive of of the aglycone as the previously reported (3β,25R)-20,22-seco- the oligoglycosidic structure [18]. Its molecular formula, 25-furost-5-en-20,22-dione-3,26-diol. The HMBC spectrum of 6

C50H80O22 was deduced from the HR-ESI-MS (m/z 1033. showed correlation between the proton signal at δ 1.07 (H-19) 5212, [M+H]+). The ESI-MS spectrum in positive-ion mode and the carbon signals at δ 140.6 (C-5), 50.1 (C-9), 36.7 (C-10), showed m/z 1055 [M+Na]+, 923 [(M+Na+)−132]+, 909 between the proton signal at δ 2.15 (H-21) and the carbon sig- [(M+Na+)−146]+, 893 [(M+Na+)−162]+, 615 [(M+ nals at δ 66.3 (C-17), 205.3 (C-20). But the HMBC correlation be- Na+)−132−146−162]+. The 1H NMR spectrum of 3 tween the proton signal at δ 2.15 (s, H-21) and the carbon signals 792 S. Shen et al. / Fitoterapia 83 (2012) 785–794 at δ 173.0 (C-22) was not observed. These data convinced the spectrum showed absorption bands at 3423, 1043 cm−1, presence one double bonds was at C-5 (6) and the E ring of 6 suggestive of the oligoglycosidic structure [18]. Its molecular was opened between C-20 and C-22. Acid hydrolysis of 6 formula, C50H82O22 was deduced from the HR-ESI-MS (m/z afforded rhamnose, glucose and xylose. Combined with its 1H 1035.5379 [M+H]+). The ESI-MS spectrum in positive-ion and 13C NMR data, the configurations of the anomeric carbons mode showed m/z 1057 [M+ Na]+, 925 [(M+Na+) − + + + + + of sugars were determined to be β-D for glucose, xylose and α-L 132] , 911 [(M+Na ) − 146] , 895 [(M+Na ) − 162] , for rhamnose [14,15]. The sugar sequences of and its linkage to 617 [(M+Na+) − 132−146− 162]+, attributable to the se- C-3 and C-26 of the aglycone were ascertained by HMBC correla- quential losses of a pentose, a deoxyhexose, and two hexose tions (Fig. 2). According to the accumulated evidence above, the residues. The 1HNMRspectrumof7 showed two methyl structure of the 6 was identified as 26-O-β-D-glucopyranosyl- singlet signals at δ 0.93, 1.06; three methyl doublet signals 3β,26-dihydroxyl-20,22-seco-(25R)-furost-5-en-20,22-dione 3- at δ 1.35 (d, J= 6.6 Hz), 0.99 (d, J =6.6 Hz) and 1.80 (d, O-β-D-xylopyranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-β- J=6.3 Hz); one olefinic proton at δ 5.28 (d, J=3.6Hz); D-glucopyranoside, named as pallidifloside H (6). four anomeric pronton signals at δ 4.97 (d, J =7.8 Hz), 6.31 Compound 7 was obtained as an amorphous white solid (s), 5.07 (d, J=7.8 Hz) and 4.84 (d, J=7.5Hz). The 13C 20 with [α]D −54.9 (MeOH, c 0.52) and it gave positive Lieber- NMR spectrum showed characteristic signals for three angu- mann–Burchard and positive Ehrlich reagent tests. The IR lar methyl group at δ 16.2 and 19.1, three secondary methyl

Fig. 2. Key HMBC correlations of compounds 1, 2, 5, 6 and 7. S. Shen et al. / Fitoterapia 83 (2012) 785–794 793

Fig. 2 (continued).

groups at δ 16.2, 17.2 and 18.5, one trisubstituted double δHb 0.33 ppm (b0.48 ppm), indicated the configuration of bonds at δ 140.4, 121.6, one hemiketal carbon signal at δ C-25 was R [14].Theα-configuration of the C-22 hydroxyl 110.4, and four anomeric carbon signals at δ 99.7, 101.7, group of the aglycone moiety was confirmed by the NOESY 105.5 and 104.7. These data indicated that 7 was a furostanol correlation between δ 2.23 (td, 6.8, 1.9, H-20) and 2.03 (m, saponin with four sugar units. A comparison of the NMR H-23). Acid hydrolysis of 7 afforded rhamnose, glucose and spectral data of 7 (Table 5) with literature data [13] allowed xylose. Combined with its 1Hand13C NMR data, the config- the identification of the aglycone as the protodioscin. The urations of the anomeric carbons of sugars were determined geminal proton resonances of H2-26 appeared at δ 3.60 to be β-D for glucose, xylose and α-L for rhamnose [14,15]. (Ha-26) and δ 3.93 (Hb-26), which showed the Δab of δHa- The sugar sequences and its linkage to C-3 and C-26 of the

Table 6 Cyotoxic activity of the isolated compounds 1–12 on C6 and Hela cell lines.

a b a b Compound C6 cell line Inhibition rate (%) IC50 (μM) Hela cell line Inhibition rate (%) IC50 (μM) 1 93.1±10.6 53.2±3.2 96.0±2.4 75.8±4.5 2 14.8±5.6 6.2±2.0 3 10.8±1.2 8.4±2.4 4 0.6±1.9 9.2±1.8 5 −2.9±0.7 12.2±0.5 6 −2.5±1.1 5.7±0.8 7 32.1±6.7 −3.6±0.7 8 −2.1±0.4 11.8±2.1 9 −5.0±2.9 15.3±4.3 10 98.3±2.7 24.1±1.7 99.9±8.0 28.1±3.9 11 98.7±7.6 10.3±2.2 92.9±3.7 9.4±1.1 12 97.8±1.65 5.1±0.2 86.9±2.3 5.2±0.9 Resveratrol 98.5±5.2 24.8±1.8 99.2±6.8 28.3±1.4 a Data are expressed as percentage of cell growth at the sample concentration of 100 μM (Data represent mean±SEM of three independent experiments). bData represent the mean±SEM of three independent experiments. 794 S. Shen et al. / Fitoterapia 83 (2012) 785–794 aglycone were ascertained by HMBC correlations (Fig. 2). References According to the accumulated evidence above, the structure of the 7 was identified as 26-O-β-D-glucopyranosyl-(25R)-5- [1] Xu DM, Huang NX, Wang SQ, Wen XG, Wu XY. Chemical constituents of – β α β → α Fritillaria pallidiflora Schrenk. Zhiwu Xuebao 1990;32(10):789 93. en-furost-3 ,20 ,26-triol 3-O- -D-xylopyranosyl(1 4)-[ -L- [2] Ruan HL, Zhang YH, Wu JZ. Advances in studies on non-alkaloid constit- rhamnopyranosyl(1→2)]-β-D-glucopyranoside, named as palli- uents of Fritillaria L. plants. Zhongcaoyao 2002;33(9):858–60. difloside I (7). [3] Furuya S, Takayama F, Mimaki Y, Sashida Y, Satoh K, Sakagami H. Cyto- toxic activity of saponins from Camassia leichtlinii against human oral To the best of our knowledge, 1, 2, 5, 6, 7 are new com- tumor cell lines. Anticancer Res 2001;21(2A):959–64. pounds, and the isolation of 3, 4, 8–12 from genus Fritillaria [4] Li H, Huang W, Wen YQ, Gong GH, Zhao QB, Yu G. Anti-thrombotic ac- is described here for the first time. tivity and chemical characterization of steroidal saponins from Dios- – All isolated steroidal saponins (1–12)weretestedforin corea zingiberensis C.H. Wright. Fitoterapia 2010;81(8):1147 56. [5] Shao B, Guo HZ, Cui YJ, Ye M, Han J, Guo DA. Steroidal saponins from vitro cytotoxicity against C6 and Hela cells. The results revealed Smilax China and their anti-inflammatory activities. Phytochemistry the furostanol saponins (4–9) and C-21 steroidal saponin (2, 3) 2007;68(5):623–30. showed no cytotoxicity (IC >100 μM), while the C-22 steroi- [6] Zhang Y, Zhang YJ, Jacob MR, Li XC, Yang CR. Steroidal saponins from 50 the stem of Yucca elephantipes. Phytochemistry 2008;69(1):264–70. dal saponin (1) and spirostanol saponins (10, 11, 12)showed [7] Yin J, Kouda K, Tezuka Y, Tran QL, Miyahara T, Chen YJ, Kadota S. Steroi- cytotoxicity against C6 and Hela cells (Table 6), when resvera- dal glycosides from the rhizomes of Dioscorea spongiosa. J Nat Prod 2003;66(5):646–50. trol used as the positive control had the IC50 value of 24.8± μ μ [8] Meng ZY, Xu SX, Li W, Sha Y. New saponins from Anemarrhena asphode- 1.8 M(C6)and28.3±1.4 M (Hela). Among the three spiros- loides Bge. Zhongguo Yaowu Huaxue Zazhi 1999;9(4):294–8. tanol saponins, compound 12 showed the strongest cytotoxic [9] Geng Y, Tan NH, Jun Zhou, Kong LY. Isolation and identification of ste- activity, compound 11 showed moderate cytotoxic activity, roid saponins from the fresh rhizomes of Dioscorea panthaica. Zhong- guo Tianran Yaowu 2004;2(1):25–7. compound 10 showed the weakest activity. Compound 11 are [10] Ono M, Takamura C, Sugita F, Masuoka C, Yoshimitsu H, Ikeda T, Nohara structurally realated to 10 without the C-17 α hydroxyl T. Two new steroid glycosides and a new sesquiterpenoid glycoside group. So, an introduction of a C-17 α hydroxyl group to the from the underground parts of Trillium kamtschaticum. Chem Pharm Bull 2007;55(4):551–6. aglycone of spirostanol saponin reduced its cytotoxic activity. [11] Hou SJ, Zou CC, Zhou L, Lei PS, Yu DQ. Synthesis of three natural dios- Compound 11 are structurally related to 12 without the termi- genyl glycosides. J Asian Nat Prod Res 2006;8(8):689–96. nal glucosyl group linked to C-4 of the inner glucosyl residue. [12] Hayes PY, Lambert LK, Lehmann R, Penman K, Kitching W. Spectral as- So, an introduction of a terminal glucosyl group linked to C-4 signments and reference data complete 1H and 13C assignments of the four major saponins from Dioscorea villosa (wild yam). Magn Reson of the inner sugar residue increased their cytotoxic activity. Chem 2007;45(11):1001–5. Compounds 1, 3, 10 and 11 process the same sugar unit. Com- [13] Ahmad VU, Khaliq-uz-Zaman SM, Shameel S, Perveen S, Ali Z. Steroidal saponins from Asparagus dumosus. Phytochemistry 1998;50(3):481–4. pounds 1, 10 and 11 showed cytotoxic activity (Table 6). But, — μ [14] Kostova I, Dinchev D. Saponins in Tribulus terrestris chemistry and compound 3 showed no cytotoxic activity (IC50 >100 M). To bioactivity. Phytochem Rev 2005;4(2–3):111–37. sum up, cytotoxic activity of the steroidal saponins is related [15] Agrawal PK, Jain DC, Gupta PK, Thakur RS. Carbon-13 NMR spectrosco- to the structures of the aglycones and the sugar unit. py of steroidal sapogenins and steroidal saponins. Phytochemistry 1985;24(11):2479–96. [16] Agrawal PK. Assigning stereo-diversity of the 27-Me group of Acknowledgements furostane-type steroidal saponins via NMR chemical shifts. Steroids 2005;70(10):715–24. [17] Yokosuka A, Mimaki Y, Sashida Y. Steroidal and pregnane glycosides This work was supported by National Key Technology from the rhizomes of Tacca chantrieri. J Nat Prod 2002;65(9):1293–8. R&D Program (2012BAI30B02); SCI-TECH R&D Program of [18] Yokosuka A, Mimaki Y, Sashida Y. Steroidal saponins from Dracaena Shihezi University (ZRKX2009ZD05). surculosa. J Nat Prod 2000;63(9):1239–43. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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