Five new furostanol saponins from the rhizomes of Tupistra chinensis

Fitoterapia 83 (2012) 323–328

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Five new furostanol saponins from the rhizomes of Tupistra chinensis Cheng-Xiong Liu, Zhi-Yong Guo, Yan-Hong Xue, Jun Cheng, Nian-Yu Huang, Yuan Zhou, Fan Cheng, Kun Zou ⁎ Hubei Key Laboratory of Natural Products Research and Development, College of Chemistry and Life Science, China Three Gorges University, Yichang 443002, PR China

a r t i c l e

i n f o

Article history: Received 10 August 2011 Received in revised form 5 November 2011 Accepted 13 November 2011 Available online 20 November 2011 Keywords: Tupistra chinesis Steroidal saponin Furostanol Diastereoisomer Cytotoxicity

a b s t r a c t Five new furostanol saponins (1–5), together with three known compounds (6–8) were obtained from the n-butanol soluble fraction of ethanol extract from Tupistra chinensis. Their structures were determined on the basis of chemical methods and spectral data. The isolated compounds were tested in vitro for their cytotoxic activities against the A549, HepG 2 and Caski cancer cell lines. Among them, compounds 6, 7, and 8 showed cytotoxicity against A549 cancer cell lines with IC50 values of 6.6, 6.7 and 29.1 μM, respectively. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Saponins are a class of natural compounds, which possess many bioactivities, such as anti-spasmodic activity [1,2], antiinflammatory, ion channel-blocking, immune-stimulating, anti-fungal [3–4], anti-fertility activity [5], and anti-tumor activity [6]. Tupistra chinensis Bak belongs to family Liliaceae (Convallarieae), which contains steroidal saponins, and it is mainly distributed in southwestern China. As a folk medicine, this species has usually been used to reduce carbuncles and ameliorate pharyngitis [7]. In the previous papers we reported the isolation and identification of 15 new furostanol saponins from the rhizomes of T. chinensis [8–14]. During the continuing screen for bioactive saponins with potential antitumor activity [15], we isolated five new furostanol saponins (1–5) (see Fig. 1) together with three known saponins from the rhizomes of T. chinensis. 1 and 2, and 3 and 4 were two pairs of diastereoisomers with the absolute configuration of C-25 for 1 and 3 being S, and that of 2 and 4 being R. To our best knowledge, only 25R isomers were isolated from the Reineckea species, and 25S isomers were obtained as the ⁎ Corresponding author. Tel./fax: + 86 717 6397478. E-mail address: [email protected] (K. Zou). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.11.010

major isomers from the Rohdea species among the Convallarieae plants [16]. In the previous literatures we reported the isolation and identification of compounds 6 and 8 [13,14], but this paper reported the cytotoxic activities of compounds 1–8 for the first time. The isolated compounds 1–8 were evaluated for in vitro cytotoxic activity against cancer cell lines A549, HepG 2 and caski using the MTT assay method, compounds 6, 7 and 8 showed cytotoxicity against A549 cancer cell lines with IC50 values of 6.6, 6.7 and 29.1 μM, respectively. But the compounds 1–5 did not show significant bioactivity against all of the above three cancer cell lines. 2. Experimental 2.1. General The melting points were measured on an X-4 digital melting point apparatus without correction. IR spectra were measured on a Nicolet FT360 instrument (San Francisco, USA) as samples in pressed KBr disks. The 1H, 13C and 2D NMR spectra were recorded on a Bruker AM-500 spectrometer with Me4Si as the intestinal standard. Mass spectra (ESI-MS, HRESI-MS) were measured in the fast atom bombardment mode using a VG AUTO Spec-300 mass spectrometer and in

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the electron spray mode using a Finnigan-MAT LCQ DECA XP plus mass spectrometer, respectively, and ions are given in m/z. HPLC was performed using a Varian ProStar 1510 system for analytical (YMC-Pack ODS-AQ column: 5 μm, 60 Å, 250 × 4.6 mm i.d.), semi preparative (YMC-Pack ODS-AQ column: 5 μm, 120 Å, 250 × 10 mm i.d.), and preparative (YMCPack ODS-AQ column: 5 μm, 120 Å, 250 × 20 mm i.d.) HPLC. Macroporous resins (AB-8, Nankai, China), RP C18 Silica Gel (100–200 mesh, YMC, Japan) were used as packing materials for column chromatography. Optical density (OD) values in the cytotoxic activity by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays were read on automated microplate reader (ELX 800, France). 2.2. Plant material The rhizomes of T. chinensis were collected from Changyang, Hubei province of China in August 2004 and identified by Professor Chen Faju. A voucher specimen (Herbarium No.: 2002ZW07408) has been deposited in the Herbarium of Department of Medicinal Plants, College of Chemistry and Life Science, China Three Gorges University, Yichang. 2.3. Extraction and isolation Air-dried powdered rhizomes (10 kg) were extracted with EtOH (30 L × 6 ) under reflux. After the removal of solvent in vacuo and freeze-drying, the EtOH extract (4000 g) was obtained. The extract was suspended in water (3.6 L), and then extracted with petroleum ether, EtOAc and nBuOH, respectively. The n-BuOH-soluble extract (600 g out of 1383 g) was dissolved in water (2.0 L), and then was subjected to macroporous resin column chromatography (10 cm × 100 cm) with gradient elution (100% water → 100% EtOH). The 70% EtOH eluate (50 g out of 300 g) was separated by Rp-C18 silica gel column chromatography (5 cm × 60 cm) in elution with gradient solvent system (100% water → 100% acetonitrile) to give 90 fractions. Fractions 34–36 (1.8 g) were further separated by repeated preparative HPLC eluted with 45% methanol (within 90 min, 4.0 mL/min, 203 nm), giving compound 1 (28 mg) and compound 2 (15 mg). Fractions 61–65 (950 mg) were further separated by repeated preparative HPLC eluted with 60% methanol (within 100 min, 4.0 mL/min, 203 nm), to afford compound 3 (40 mg) and compound 4 (35 mg). Fractions 52–53 (500 mg) were further separated by repeated preparative HPLC eluted with gradient solvent (15% → 25% acetonitrile, within 60 min, 4.0 mL/min, 203 nm), and then repeated semi preparative HPLC eluted with gradient solvent (20% → 25% acetonitrile, within 30 min, 2.5 mL/min, 203 nm), compound 5 (30 mg) was obtained. Fractions 8–12 (1.0 g) were further separated by repeated preparative HPLC eluted with 30% methanol (within 90 min, 4.0 mL/min, 203 nm), giving compound 6 (15 mg) and compound 7 (20 mg). Fractions 15–16 (300 mg) were further separated by repeated preparative HPLC eluted with 35% methanol (within 90 min, 4.0 mL/min, 203 nm), giving compound 8 (22 mg). Compound 1, white amorphous powder, [α]D20-63.3° (c =0.52; CH3OH); IR (KBr) cm− 1: 3429, 2926, 1632, 1560; the data for 1H NMR (500 MHz, Pyridine-d5) δ: 3.91 (H-1, br d,

J =8.0 Hz), 2.34 (H-2, br d, J = 13 Hz), 4.60 (H-3, br s), 2.48 (H-17, d, J = 10.5 Hz), 0.72 (H-18, s), 1.28 (H-19, s), 1.64 (H21, s), 3.50 (H-26b, dd, J =7.0, 16.0 Hz), 4.08 (H-26a, dd, J =5.5, 15 Hz), 1.05 (H-27, d, J =6.5 Hz), 4.84 (H-1′, d, J =7.5 Hz), 5.01 (H-1″, d, J =7.5 Hz); 13C NMR (125 MHz, Pyridine-d5) see Table 1; ESI-MS (positive ion mode) m/z: 795.6 [M+ K]+, 779.6 [M+Na]+, 757.6 [M+H]+, 595.5 [M-Glc+ H]+; HR-ESI-MS (positive ion mode) m/z: 779.4194 [M+Na]+ (calcd. for C39H64NaO14, 779.4199). Compound 2, white amorphous powder, [α]D20-26.6° (c= 0.49; CH3OH); IR (KBr) cm− 1: 3425, 2915, 1600, 1560; the data for 1H NMR (500 MHz, Pyridine-d5) δ: 3.91 (H-1, br d , J = 8.0 Hz), 2.34 (H-2, br d, J = 13 Hz), 4.62 (H-3, br s), 2.49 (H-17, d, J = 10.5 Hz), 0.72 (H-18, s), 1.28 (H-19, s), 1.65 (H-21, s), 3.64 (H-26b, dd, J = 9.5, 15.0 Hz), 3.97 (H-26a, dd, J = 9.5, 16.5 Hz), 1.04 (H-27, d, J = 6.5 Hz), 4.87 (H-1′, d, J = 8.0 Hz), 5.03 (H-1″, d, J = 8.0 Hz); 13C NMR (125 MHz, Pyridine-d5) see Table 1; ESI-MS (positive ion mode) m/z:

Table 1 13 C NMR spectral data of compounds 1–5 (125 MHz in Pyridine-d5). Position

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 3-O-glc-1 2 3 4 5 6 26-O-glc-1 2 3 4 5 6 (1 → 4)3-O-glc-1 2 3 4 5 6

72.4 32.0 75.1 29.3 31.0 26.6 26.6 35.5 42.1 40.4 21.2 40.1 43.6 54.7 34.5 84.5 64.7 14.5 19.1 103.6 11.8 152.5 31.4 23.7 33.7 75.3 17.2 101.5 75.3 78.8 71.8 78.5 62.8 105.2 74.8 78.7 71.7 78.6 62.9

72.4 32.0 75.2 29.3 31.0 26.6 26.6 35.5 42.1 40.4 21.2 40.0 43.6 54.6 34.5 84.5 64.7 14.5 19.1 103.6 11.8 152.4 31.5 23.7 33.5 75.3 17.4 101.4 75.3 78.7 71.8 78.6 62.8 104.9 74.8 78.7 71.7 78.6 62.8

72.4 32.0 75.3 29.3 31.0 26.6 26.6 35.5 42.1 40.4 21.3 40.1 43.6 54.7 34.5 84.5 64.9 14.5 19.2 105.6 11.8 152.5 31.5 23.7 33.8 74.8 17.2 105.2 74.6 76.9 81.3 76.8 62.5 101.0 75.3 78.6 71.6 78.5 62.1 105.0 74.9 78.7 71.8 78.3 62.9

72.4 33.5 74.8 29.3 31.0 26.5 26.6 35.5 42.1 40.4 21.2 40.0 43.6 54.6 34.5 84.5 64.7 14.5 19.1 104.9 11.8 152.2 31.5 23.7 33.7 74.2 17.4 105.2 75.0 76.9 81.3 76.8 62.5 101.0 75.2 78.5 71.6 78.5 62.1 105.0 74.9 78.6 71.8 78.3 62.9

74.9 33.5 73.9 38.9 139.8 124.3 367 32.4 50.6 44.1 23.7 40.4 43.1 55.3 32.4 84.4 64.7 14.4 13.3 103.7 11.8 152.3 31.4 24.7 34.6 74.9 17.3 102.9 74.8 78.6 71.7 78.4 62.9 104.9 75.0 78.7 71.8 78.5 62.9

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795.7 [M+ K]+, 779.7 [M+ Na]+, 757.7 [M+ H]+, 595.6 [MGlc +H]+; HR-ESI-MS (positive ion mode) m/z: 757.4367 [M+ H]+ (calcd. for C39H65O14, 757.4374). Compound 3, white amorphous powder, [α]D20-43.4° (c = 0.38; 30% acetonitrile); IR (KBr) cm − 1: 3419, 2926, 1632, 1560; the data for 1H NMR (500 MHz, Pyridine-d5) δ: 3.90 (H-1, br d, J = 9.0 Hz), 2.48 (H-17, d, J = 10.0 Hz), 0.72 (H-18, s), 1.28 (H-19, s), 1.64 (H-21, s), 3.50 (H-26b, dd, J = 9.5, 16.5 Hz), 4.08 (H-26a, dd, J = 9.0, 15 Hz), 1.05 (H-27, d, J = 7.0 Hz), 4.97 (H-1′, d, J = 8.0 Hz), 4.85 (H-1″, d, J = 9.5 Hz), 5.21 (H-1′′′, d, J = 8.0 Hz); 13C NMR (125 MHz, Pyridine-d5) see Table 1; ESI-MS (positive ion mode) m/z: 957.8 [M + K] +, 941.8 [M + Na] +, 919.9 [M + H] +, 757.7 [M-Glc + H] +, 595.6 [M-2Glc + H] +; HR-ESI-MS (positive ion mode) m/z: 919.4885 [M + H] + (calcd. for C45H75O19, 919.4903). Compound 4, white amorphous powder, [α]D20-28.5° (c = 0.23; 30% acetonitrile); IR (KBr) cm − 1: 3429, 2922, 1600, 1561; the data for 1H NMR (500 MHz, Pyridine-d5) δ: 3.88 (H-1, br d, J = 9.0 Hz), 2.46 (H-17, d, J = 10.0 Hz), 0.71 (H-18, s), 1.26 (H-19, s), 1.62 (H-21, s), 3.62 (H-26b, dd, J = 9.5, 16.5 Hz), 3.95 (H-26a, dd, J = 9.0, 15 Hz), 1.02 (H-27, d, J = 7.0 Hz), 4.95 (H-1′, d, J = 7.5 Hz), 4.82 (H-1″, d, J = 9.0 Hz), 5.18 (H-1′′′, d, J = 7.5 Hz); 13C NMR (125 MHz, Pyridine-d5) see Table 1; ESI-MS (positive ion mode) m/z: 941.7 [M + Na] +, 919.7 [M + H] +, 757.6 [M-Glc + H] +; HR-

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ESI-MS (positive ion mode) m/z: 919.4912 [M + H] + (calcd. for C45H75O19, 919.4903). Compound 5, white amorphous powder, [α]D20-33.4° (c = 0.40; CH3OH); IR (KBr) cm − 1: 3429, 2916, 1601, 1560; the data for 1H NMR (500 MHz, Pyridine-d5) δ: 4.47 (H-1, m), 0.78 (H-18, s), 1.28 (H-19, s), 1.59 (H-21, s), 3.60 (H26b, dd, J = 9.5, 15.0 Hz), 3.92 (H-26a, dd, J = 9.5, 16.5 Hz), 1.00 (H-27, d, J = 7.0 Hz), 4.81 (H-1′, d, J = 8.5 Hz), 4.93 (H1″, d, J = 7.5 Hz); 13C NMR (125 MHz, Pyridine-d5) see Table 1; ESI-MS (positive ion mode) m/z: 777.6 [M + Na] +, 755.5 [M + H] +, 593.5 [M-Glc + H] +; HR-ESI-MS (positive ion mode) m/z: 755.4222 [M + H] + (calcd. for C39H63O14, 755.4218). 2.4. Cytotoxicity assay Cytotoxicity was determined by MTT assay according to a previous method [17,18]. Briefly, cells were incubated with different concentration samples (n = 12) (50, 25 to 1.56 μg/ mL). Controls with non-treated cells were run in parallel (n = 12). Following a 48 h incubation period, the medium was removed and the cells were further incubated for 1 h in the presence of 50 μL of MTT (0.7 mg/mL in phosphate buffered saline) (20 μL MTT (5 mg/mL in phosphate buffered saline) was added, and the cells were further incubated for 4 h). After dissolution of the resulting crystal formazan by

Fig. 1. The structures of compounds 1–8.

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the addition of DMSO (150 μL), the absorbance (A) was measured at 490 nm using an automated microplate reader. The inhibitory rate is calculated as [(1 − At / Ac) × 100], where At and Ac are the mean absorbance values of the n wells containing TST (t) and control cells without treatment (c), respectively. 3. Results and discussion Compound 1 was obtained as a white amorphous powder, which showed positive reaction in the Liebermann–Burchard and Molish test. The high resolution-electrospray ionizationmass spectrometry (HR-ESI-MS) showed a quasi-molecular ion peak at m/z 779.4194 [M+ Na] + (calcd. for C39H64NaO14, 779.4199). The 1H NMR spectrum of 1 showed four methyl signals at δ 0.73 (3H, s), 1.05 (3H, d, J = 6.5 Hz), 1.28 (3H, s) and 1.64 (3H, s), and two anomeric proton signals of sugar at δ 4.84 (1H, d, J = 7.5 Hz) and 5.01 (1H, d , J = 7.5 Hz). When 1 was hydrolyzed with 2.0 M HCl, only glucose was detected in the hydrolysate on TLC and PC. Among the 39 carbon signals in the 13C NMR spectrum, 12 carbon signals were assigned to glucose, the remaining 27 signals were assignable to the aglycone, including four signals due to methyl groups at δ 14.5

(C-18), 19.1 (C-19), 11.8 (C-21), and 17.2 (C-27) and three signals due to oxygenated carbons at δ 72.4, 75.1, and 75.3. A downfield signal at δ 84.5 could be assigned to the oxygenated carbon (C-16), which located at furan ring of aglycone. The methine carbon signal at δ 64.7 could be assigned to C-17 of aglycone, The above results suggested 1 was a steroidal saponin. Two olefinic carbon signals appeared at δ 152.5 and 103.6 respectively in 13C NMR spectrum, the HMBC spectrum showed the correlation between δ 1.64 (3H, s) (H-21) and δ 103.6, δ 1.88 (H-23) and δ 152.5. So it confirmed the existence of a double bond between C-20 and C-22 in compound 1. The 5β-H configuration was revealed by a group of carbon signals due to C-4, C-5, C-6, C-7, C-8, C-9 and C-19 at δ 29.3, 31.0, 26.6, 26.6, 35.5, 42.1 and 19.2 [19]. The 25S configuration was suggested by the chemical shift value of H-27, which is larger than 1.00, and the difference of chemical shifts between H-26a at δ 4.08 and H-26b at δ 3.50, Δab (δa − δb) being larger than 0.57 [20]. The COSY spectrum showed the proton signal δ 4.60 correlation with H-2 at δ 2.34 and H-4 at 1.90, the proton signal δ 4.60 further confirmed correlation with carbon signal δ 75.1 according to the HSQC spectrum. So the carbon signal at δ 75.1 could be

Fig. 2. The key HMBC and 1H–1H COSY correlations of compound 1, 3 and 5.

C.-X. Liu et al. / Fitoterapia 83 (2012) 323–328

assigned to C-3 based on COSY and HSQC spectra. The configuration of hydroxyl group at C-3 was deduced as β orientation from its proton chemical shift at 4.60 (br. s). The COSY spectrum of aglycone of 1 showed correlation between the proton signal of 1-OH at δ 4.45 (1H, d, J = 9.0 Hz) and 1-H at δ 3.90 (1H, br.d, J = 8.0 Hz). The comparison the 13C NMR spectral data of 1 with those of a reference compound, furost-1β, 3β, 22α, and 26-tetraol [10], we found that the carbon signals of compound 1 in the A–F rings were almost identical, except for C-20–C-27 due to the presence of double bond between C-20 and C-22. Thus, the aglycone of compound 1 was determined as furost-20(22)-en-1β, 3β, 26-triol. Two anomeric proton signals of sugars were observed at δ 4.84 (1H, d, J = 7.5 Hz) and 5.01 (1H, d, J = 7.5 Hz) in the 1H NMR spectrum of 1, showing that the two sugars had β-configurations, and the correlated carbon signals were observed at δ 105.2 and 101.5 in the HSQC spectrum of 1, respectively. The HMBC spectrum of 1 displayed the correlations between δ 4.84 (Glc H-1) and δ 75.3 (C-26), indicating that the glucose moiety was attached to C-26 of aglycone. Furthermore, the glycosylation of C-3 hydroxyl group was revealed by the cross peaks between δ 5.01 (Glc′) and δ 75.1(C-3) in the HMBC spectrum of 1. Thus, 1 was identified as (25S)-26-O-β-D-glucopyranosyl-furost-1β, 3β, 26trihydroxy-20(22)-en-3-O-β-D-glucopyranoside(see Table 1 and Fig. 2). Compound 2 was obtained as a white amorphous powder. It appeared closely behind but distinctly different from compound 1, their retention times were 29.3 min (compound 1) and 29.8 min (compound 2) respectively in the HPLC chromatogram. The HR-ESI-MS results of compounds 1 and 2 displayed same quasi-molecular ion peak at m/z 757.4367 [M + H] + (calcd. for C39H65O14, 757.4374). Compounds 1 and 2 showed identical 1H and 13C NMR spectra, except for proton signals due to H-27 and H-26 of the aglycone. The 25R configuration was suggested by the chemical shift value of H-27, which is less than 1.05, and the difference of chemical shifts between H-26a δ 3.97 and H-26b δ 3.64, Δab (δa − δb) being smaller than 0.48 [20]. Apparently, the compounds 1 and 2 were a pair of diastereoisomers. Therefore, 2 was identified as (25R)-26-O-β-D-glucopyranosyl-furost-1β,3β,26-trihydroxy-20(22)-en-3-O-β-D-glucopyranoside . Compound 3 was obtained as a white amorphous powder, which showed positive reaction in the Liebermann–Burchard and Molish test. The high resolution-electrospray ionizationmass spectrometry (HR-ESI-MS) showed a quasi-molecular ion peak at m/z 919.4885 [M + H] + (calcd. for C45H75O19, 919.4903). The 1H NMR spectrum of 3 showed the presence of four methyl signals at δ 0.73 (3H, s), 1.05 (3H, d, J = 7.0 Hz), 1.28 (3H, s) and 1.64 (3H, s), and three anomeric proton signals of sugars at δ 4.85 (1H, d, J = 9.5 Hz), 4.97 (1H, d, J = 8.0 Hz) and 5.21 (1H, d, J = 8.0 Hz). When 3 was hydrolyzed with 2.0 M HCl, only glucose was detected in the hydrolysate on TLC and PC. Among the 45 carbon signals in the 13C NMR spectrum, 18 carbon signals were assigned to glucose, the remaining 27 signals were assignable to the aglycone, including four signals due to methyl groups at δ 14.5 (C-18), 19.2 (C-19), 11.8 (C-21), and 17.2 (C-27) and three signals due to oxygenated carbons at δ 72.4, 75.3, and 74.8. The above results suggested 3 was a steroidal saponin. The comparison of the 13C NMR spectral data of the aglycone in

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compound 3 with the aglycone in compound 1 showed that their chemical shifts were in good agreement, the structural difference between compounds 3 and 1 was only the different glucosyl residue moieties, compound 1 contained two glucosyl residues and compound 3 encompassed three glucosyl residues. The 25S configuration was suggested by the chemical shift value of H-27, which is larger than 1.00, and the difference of chemical shifts between H-26a at δ 4.08 and H-26b at δ 3.50, Δab (δa − δb) being larger than 0.57 [20]. Three anomeric proton signals of sugars were observed at δ 4.85 (1H, d, J = 9.5 Hz), 4.97 (1H, d, J = 8.0 Hz) and 5.21 (1H, d, J = 8.0 Hz) in the 1H NMR spectrum of 3, showing that the three sugars had β-configuration and the correlated carbon signals were observed at δ 105.2, 101.0 and 105.0 in the HSQC spectrum of 3, respectively. The HMBC spectrum of 3 displayed the δ 4.85 (Glc H-1) and δ 74.8 (C-26), indicating that the glucose moiety was attached to C-26 of aglycone. Furthermore, the glycosylation of C-3 hydroxyl group was revealed by the cross peaks between δ 4.97 (Glc′ H-1) and δ 75.3 (C-3) in the HMBC spectrum of 3. Finally, a 1 → 4 linkage of sugar moieties at C-3 was determined by the HMBC data of 3, δ 5.21 (Glc″ H-1) correlated with δ 81.3 (Glc′ C-4), δ 4.40 (Glc′ H-4) correlated with δ 105.0 (Glc″ C-1). Thus, 3 was identified as (25S) -3-O-β-Dglucopyranosyl-(1 → 4)-β-D-glucopyranosyl-furost-1β, 3β, 26-trihydroxy-20(22)-en-26-Oβ-D-glucopyranoside (see Table 1 and Fig. 2). Compound 4 was obtained as a white amorphous powder. It appeared closely behind but distinctly different from compound 3, their retention times were 25.5 min (compound 3) and 26.2 min (compound 4) respectively in the HPLC chromatogram. Compounds 4 and 3 gave same quasi-molecular ion peak at m/z 919.4912 [M + H] + (calcd. for C45H75O19, 919.4903). Compounds 3 and 4 showed almost identical 1H and 13C NMR spectra, except for proton signals due to H-27 and H-26 of the aglycone. The 25R configuration was suggested by the chemical shift value of H-27, which is less than 1.05, and the difference of chemical shifts between H26a δ 3.95 and H-26b δ 3.62, Δab (δa − δb) being smaller than 0.48 [20]. Apparently, the compounds 3 and 4 were a pair of diastereoisomers. Therefore, 4 was identified as (25R)-3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosylfurost-1β, 3β, 26-trihydroxy-20(22)-en-26-O-β-Dglucopyranoside. Compound 5 was obtained as a white amorphous powder, which showed positive reaction in the Liebermann–Burchard and Molish test. The high resolution-electrospray ionizationmass spectrometry (HR-ESI-MS) showed a quasi-molecular ion peak at m/z 755.4222 [M + H] + (calcd. for C39H63O14, 755.4218). The 1H NMR spectrum showed the presence of four methyl groups at δ 0.78 (3H, s), 1.00 (3H, d, J = 7.0 Hz) and 1.28 (3H, s), 1.59 (3H, s) and two anomeric proton signal of sugar at δ 4.81 (1H, d, J = 8.5 Hz), 4.93 (1H, d, J = 7.5 Hz). When 5 was hydrolyzed with 2.0 M HCl, only glucose was detected in the hydrolysate on TLC and PC. The 13C NMR spectrum exhibited 27 carbon signals arising from the aglycone moiety, the remaining 12 signals were assignable to glucose, including four signals due to methyl groups at δ 14.4 (C18), 13.3 (C-19), 11.8 (C-21), and 17.3 (C-27), three signals due to oxygenated carbons at δ 74.9, 73.9, and 74.9 and two anomeric carbon signals at δ 104.9 and 102.9. The above results suggested that 5 was a steroidal saponin with two

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glucosyl residues. The comparison of the 1H and 13C NMR spectra of 5 and 1 shows that, they were almost identical except the signal of C-4–C-7 and C-19, and there were two additional carbon signals at δ 139.8 and 124.3 in the carbon spectrum of compound 5, implying the existence of a double bond in the A or B ring. The HMBC spectrum showed the correlations between the proton signal of H-19 at δ 1.28 (3H, s) and the carbon signals δ 139.8; the olefinic bond proton signal δ 5.63 (1H, d, J = 4.5 Hz) and the three carbon signals δ 32.4 (C-4), 38.9 (C-8), and 44.1 (C-10) indicating that another olefinic bond was linked between C-5 and C-6. In the HMBC spectrum of compound 5, the correlations of δ 4.93 (1H, d, J = 7.5 Hz, Glc′ H-1) to δ 73.9 (C-3), and δ 4.81 (1H, d, J = 8.5 Hz, Glc H-1) to δ 74.9 (C-26), displayed that the two sugar moieties were connected to the aglycone at the 3- and 26-positions. The 25R configuration was suggested by the chemical shift value of H-27, which is less than 1.05, and the difference of chemical shifts between H-26a δ 3.92 and H-26b δ 3.60, Δab (δa − δb) being smaller than 0.48 [20]. Finally, the structure of 5 was determined as (25R) -26-O-β-D-glucopyranosyl-furost-1β,3β,26-trihydroxy-5 (6),20(22)-dien-3-O-β-D-glucopyranoside (see Table 1 and Fig. 2). All the isolated compounds (1–8) were evaluated for in vitro cytotoxic activity against cancer cell lines A549, HepG 2 and Caski using the MTT assay method, compounds 6, 7 and 8 showed cytotoxicity against A549 cancer cell lines with IC50 values of 6.6, 6.7 and 29.1 μM, respectively. But the compounds 1–5 did not show bioactivity against all of these three cell lines. Comparison of the cytotoxic activities and structures of compounds 1–8 suggested that the double bond at C20–C22 position decreased their cytotoxic activities. Acknowledgments This research was financially supported by the National Natural Science Foundations of China (Nos. 30870254, 31070313 and 30670213), Natural Science Foundation of Hubei Province (No. 2007ABC008). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.fitote.2011.11.010.

References [1] Barile E, Capasso R, Izzo AA, Lanzotti V, Sajjadi SE, Zolfaghari B. Structure–activity relationships for saponins from Allium hirtifolium and Allium elburzense and their antispasmodic activity. Planta Med 2005;71: 1010–8. [2] Corea G, Fattorusso E, Lanzotti V, Capasso R, Izzo AA. Antispasmodic saponins from bulbs of red onion, Allium cepa L Var. Tropea. J Agric Food Chem 2005;53:935–40. [3] Lanzotti V. Bioactive saponins from Allium and Aster plants. Phytochem Rev 2005;4:95–110. [4] Wang Xiankai. Natural medicine chemistry. Beijing: People's medical publishing house; 1988. [5] Pant G, Panwar MS, Negi DS, Rawat MSM, Morris GA. Spirostanol glycoside from fruits of Asparagus officinalis. Phytochemistry 1988;27:3324–5. [6] Sparg SG, Light ME, Staden J. Biological activities and distribution of plant saponins. J Ethnopharmacol 2004;94:219–43. [7] Zhan YH. China Shenongjia resources of medicinal plants. Wuhan: Hubei Scientific and Technological Press; 1994. [8] Zou K, Wu J, Liu C, Xiao ZH. A furostanol saponin from the cytotoxic fraction of Tupistra chinensis rhizomes. Chin Chem Lett 2006;17:1335–8. [9] Zou K, Wang JZ, Du M, Li Q, Tu GZ. A pair of diastereoisomeric steroidal saponins from cytotoxic extracts of Tupistra chinensis rhizomes. Chem Pharm Bull 2006;54:1440–2. [10] Zou K, Wu J, Du M, liu C, Tu GZ, Wang JZ. Diastereoisomeric saponins from the rhizomes of Tupistra chinensis. Chin Chem Lett 2007;18:65–8. [11] Zou K, Wang JZ, Wu J, Zhou Y, Liu C, Dan FJ, et al. Furostanol saponins with inhibitory action against COX-2 production from Tupistra chinensis rhizomes. Chin Chem Lett 2007;18:1239–42. [12] Xu LL, Zou K, Wang JZ, Wu J, Zhou Y, Dan FJ, et al. New polyhydroxylated furostanol saponins with inhibitory action against NO production from Tupistra chinensis rhizomes. Molecules 2007;12: 2029–37. [13] Zou K, Wang JZ, Guo ZY, Du M, Wu J, Zhou Y, et al. Structural elucidation of four new furostanol saponins from Tupistra chinensis by 1D and 2D NMR spectroscopy. Magn Reson Chem 2009;47:87–91. [14] Guo ZY, Zou K, Wang JZ, Liu C, Tang ZC, Yang CY. Structural elucidation and NMR spectral assignment of three new furostanol saponins from the roots of Tupistra chinensis. Magn Reson Chem 2009;47:613–6. [15] Kostova I, Dinchev D. Saponins in Tribulus terrestris—chemistry and bioactivity. Phytochem Rev 2005;4:111–37. [16] Chen MJ. Steroidal glycosides from convallarieae. Chin Bull Bot 1999;16: 37–43. [17] Chen TK, Ales DC, Baenziger NC, Wiemer DF. Anti-repellent triterpenoids from Cordia alliodora. J Org Chem 1983;48:3525–31. [18] Keawpradub N, Eno-Amooquaye E, Burke PJ, Houghton PJ. Cytotoxic activity of indole alkaloids from Alstonia macrophylla. Planta Med 1999;65:311–5. [19] Agrawal PK, Dharam Jain C, Pathak Ashish K. NMR spectroscopy of steroidal sapogenins and steroidal saponins: an update. Magn Reson Chem 1995;33:923–53. [20] Agrawal PK. Dependence of 1H NMR chemical shifts of geminal protons of glycosyloxy methylene (H2-26) on the orientation of the 27-methyl group of furostane-type steroidal saponins. Magn Reson Chem 2004;42:990–3.