Two new cytotoxic triterpenoid saponins from the roots of Clematis argentilucida

Fitoterapia 83 (2012) 759–764

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Two new cytotoxic triterpenoid saponins from the roots of Clematis argentilucida Wenli Hai a, Hua Cheng b, Mei Zhao c, Yi Wang a, Liangjian Hong a, Haifeng Tang a,⁎, Xiangrong Tian a, d,⁎⁎ a b c d

Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, PR China Department of Pharmacy, Xi'an Children's Hospital, 69 Xijuyuan Rd., Xi'an 710002, PR China Department of Pharmacy, Tangdou Hospital, Fourth Military Medical University, Xi'an 710038, PR China Research & Development Center of Biorational Pesticide, College of Plant Protection, Northwest A & F University, Yangling, 712100, PR China

a r t i c l e

i n f o

Article history: Received 22 December 2011 Accepted in revised form 27 February 2012 Available online 11 March 2012 Keywords: Ranunculacea Clematis argentilucida Triterpenoid saponin Cytotoxicity

a b s t r a c t Bioassay-guided fractionation of the n-BuOH extract of the roots of Clematis argentilucida led to the isolation of two new triterpenoid saponins along with a known one, cussonside B (3). By extensive spectral analysis and chemical evidences, the structures of the two new saponins were determined to be 3β-O-[β-D-ribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-Larabinopyranosyl] hederagenin-11,13-dien-28-oic acid (1) and 3β-O-{β-D-ribopyranosyl(1→ 3)-α-L-rhamnopyranosyl-(1→ 2)-[β-D-glucopyranosyl-(1→ 4)]-β-D-xylopyranosyl} oleanolic acid (2), respectively. Saponin 1 is the first example of triterpenoid saponins with two double bonds located at C-11 and C-13 in the aglycone from the genus Clematis. The two new saponins exhibited significant cytotoxicity against human leukemia HL-60 cell lines, human hepatocellular carcinoma Hep-G2 cell lines and human glioblastoma U251MG cell lines with a range of IC50 values from 2.74 to 25.40 μM, while 3 showed inactivity against all of the three cancer cell lines. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The genus Clematis L. (Ranunculacea) consists of 355 species, which are widespread throughout the world. There are about 150 species (93 endemic ones) distributed in China [1]. Previous chemical and pharmacological studies have shown that triterpenoid saponins are the primary components in the

⁎ Correspondence to: H. Tang, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, 15 Changle West Rd., Xi'an 710032, PR China. Tel./fax.: + 86 29 84775471. ⁎⁎ Correspondence to: X. Tian, Research & Development Center of Biorational Pesticide, College of Plant Protection, Northwest A & F University, 22 Xinong Rd., Yangling, 712100, PR China. E-mail addresses: [email protected] (H. Tang), [email protected] (X. Tian). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.03.003

genus Clematis, these saponins have been reported to have a wide spectrum of biological effects, including various analgesic, diuretic, antitumor, antimicrobial and anti-inflammatory activities [2–5]. Clematis argentilucida is a perennial woody climber widely distributed in China, having the effect of analgesic, detoxification, and activating blood circulation. The rhizome of the plant has been used as traditional folk medicines for the treatment of wound, rheumatism, arthralgia, aphonia hoarseness as well as limb numbness [6]. In the preceding paper, our research team reported the isolation and structural elucidation of eleven triterpenoid saponins from C. argentilucida [7,8]. However, no pharmacological studies have been reported to date. As a follow-up research of exploring new bioactive compounds from natural sources, we investigated the n-BuOH extract of the roots of C. argentilucida again by bioassayguided fractionation based on the cytotoxic activities against human glioblastoma U251MG cell lines. We report herein the

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30

12 13 25 2 3

OH O O OH

24

1

10 5

4

11 26 14 9

H 6

8 7

19 18

15

O

CH3 O

O

17

16

21 22

COOH

H CH 2OH

O

23

O

OH

OH

OH O CH 3

1

Rib

OH OH OH

O

OH

OH

2

Glc Rha

OH

OH OO

Rib

O

OH O

O

OH

OH OH OH

O

Xyl

O Glc

OH

HO

O

OH O OH

Rha

O

COOH

28

27

Ara

OH

20

O

CH3 2

O

OH

OH

Glc1

OH

Rha

OH OH

3

Fig. 1. Structures of compounds 1–3 from Clematis argentilucida.

isolation, structure elucidation and biological activity of two new triterpenoid saponins (1 and 2) and a known saponin 3 obtained through the above procedure (Fig. 1). 2. Experimental procedure 2.1. General Melting points were determined on an XT5-XMT apparatus and uncorrected. Optical rotation was measured on a PerkinElmer 343 polarimeter. NMR spectra were recorded on a Bruker AVANCE-500 spectrometer in pyridine-d5, with TMS as the internal standard. ESI-MS and HR-ESI-MS were recorded on a Micromass Quattro mass spectrometer. GC were performed on a SHIMADZU GC-2010 apparatus using an LChirasil-Val column (0.32 mm × 25 m; column temperature: 100–180 °C, rate 5 °C/min; column head pressure: 12 Pa; carrier gas: He). HPLC was carried out on a Dionex P680 liquid chromatograph equipped with a UVD 170 UV/vis detector using a YMC-Pack R&D ODS-A column (5 μm, 250 mm × 20 mm i.d.) and monitored at 206 nm for the semipreparation, and a Thermo ODS-2 column (250 mm× 4.6 mm i.d.) for analysis. Chromatographic materials were performed on silica gel (10–40 μm; Qingdao Marine Chemical, Inc.), Sephadex LH-20 (GE-Healthcare) and reversed-phase Si gel (Lichroprep RP-18, 40–63 μm, Merck Inc.), respectively. Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 20% H2SO4 in EtOH (v/v). 2.2. Plant material The air-dried roots of C. argentilucida were collected from Tsinling Mountains, Shaanxi Province of China in September 2009, and were identified by Prof. Ji-Tao Wang (Department of Pharmacognosy, School of Pharmacy, Shaanxi University of Chinese Medicine). A voucher specimen (No: 20090902) was deposited in the Department of Pharmacy, Xijing Hospital, Fourth military Medical University, Xi'an, China.

2.3. Extraction and isolation The air-dried roots of C. argentilucida (5 kg) were powdered and extracted with 70% EtOH (5 L × 3, 2 h/time) under reflux. The EtOH extract was concentrated under reduced pressure to give a residue (750 g), which was suspended in water (5 L) and then partitioned successively with petroleum ether (5 L × 2) and n-BuOH saturated by H2O (5 L × 4). The n-BuOH phase was evaporated under reduced pressure to give a dark gummy residue (250 g) which was shown to be cytotoxicity against U251MG cells (IC50 = 39 μg/mL). One hundred grams of the n-BuOH extract was subjected to column chromatography over silica gel (2000 g, 15 × 120 cm) eluting with a CHCl3– MeOH–H2O gradient (1:0:0 to 6.5:3.5:1, lower phase) to give fourteen major fractions (Fr. 1–Fr. 14) based on TLC analysis. As Fr. 8, 10 and 11 showed more cytotoxic activities against U251MG cells (IC50 = 19 μg/mL, 27 μg/mL, 22 μg/mL, respectively) than the remaining eleven fractions, the isolation work was mainly focused on these three fractions. Fr. 8 (4.4 g) was chromatographed on silica gel (80 g, 4 × 70 cm) with a CHCl3– MeOH–H2O gradient (12:1:0.5 to 6.5:3.5:1) to give seven subfractions (Fr. 8–1 to Fr. 8–7). Fr. 8–6 (450 mg, IC50 = 7 μg/mL) was subjected to size exclusion chromatography on a Sephadex LH-20 column equilibrated with CHCl3–MeOH (1:1), and was further purified by repeated semipreparative HPLC (UV detection at 206 nm) to afford pure glycoside 1 (7.2 mg, MeOH– H2O, 70:30, 8 mL/min, tR = 21.5 min). Fr. 11 (0.8 g) was chromatographed on Sephadex LH-20 column equilibrated with CHCl3–MeOH (1:1) to remove pigments, and then was further purified by semipreparative HPLC (UV detection at 206 nm) to afford pure glycoside 2 (7.1 mg, MeOH–H2O, 70:30, 8 mL/ min, tR = 31.4 min). Fr. 10 (0.4 g) was chromatographed on reversed-phase Si gel silica gel eluting with MeOH–H2O gradient (40:60, 75:25) to give 2 subfractions (Fr. 10–1 and Fr. 10–2). Fr. 10–2 (350 mg) mainly contained glycosides based on Liebermann–Burchard and Molish tests, and was further purified by repeated semipreparative HPLC (UV detection at 206 nm) to afford pure glycoside 3 (13.5 mg, MeOH–H2O,

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2.4. Acidic hydrolysis of compounds 1 and 2

70:30, 8 mL/min, tR = 40 min). The purity of all compounds was assessed by HPLC as more than 95%.

Each saponin (3 mg) was heated in an ampule with 2 mol/L CF3COOH (3 mL) at 120 °C for 2 h. The reaction mixture was poured into CHCl3–H2O (1:1). The aqueous phase was evaporated under vacuo, 1 mL pyridine and 2 mg NH2OH·HCl were added to the dried residue, and the mixture were stirred at 90 °C for 1 h. After the reaction mixtures were cooled, 1.5 mL of Ac2O was added and the mixtures were heated at 90 °C for 1 h. The reaction mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC. The carbohydrates were determined by comparing the retention times with standard aldononitrile peracetates prepared from authentic sugars by the same procedure performed for the sample. The L-arabinose (Ara), L-rhamnose (Rha), and D-ribose (Rib) in a ratio of 1:1:1 for saponin 1, whereas D-xylose (Xyl), L-rhamnose, D-ribose and D-glucose (Glc) were identified in a ratio of 1:1:1:1 for saponin 2.

2.3.1. 3β-O-[β-D-ribopyranosyl-(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl] hederagenin-11,13-dien-28-oic acid (1) White amorphous powder, mp 253–256 °C, [α]D22 = −23.5 (c = 0.01, MeOH), UV λmax (241.1, 249.2 and 258.3 nm); 1H (500 MHz, pyridine-d5) and 13C (125 MHz, pyridine-d5) NMR data see Table 1; Key HMBC and NOESY correlations see Fig. 2; ESI-MS (positive ion mode) m/z 903 [M+ Na] +, ESI-MS (negative ion mode) m/z 915 [M+ Cl] −, HR-ESI-MS (positive ion mode) m/z 903.4927 [M+ Na] + (calcd. for C46H72O16Na 903.4925).

2.3.2. 3β-O-{β-D-ribopyranosyl-(1→3)-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-xylopyranosyl} oleanolic acid (2) White amorphous powder, mp 244–245 °C, [α]D22 = −21.1 (c = 0.09, MeOH); 1H (500 MHz, pyridine-d5) and 13C (125 MHz, pyridine-d5) NMR data see Table 1; Key HMBC and NOESY correlations see Fig. 2; ESI-MS (positive ion mode) m/z 1051 [M+ Na] +, HR-ESI-MS (positive ion mode) m/z 1051.5461 [M+ Na] + (calcd. for C52H84O20Na 1051.5454).

Table 1 1 H and 13C NMR data for compounds 1–2 (C5D5N, 1H NMR 500 MHz, 1

2.5. Cytotoxicity assay in vitro The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used for in vitro evaluation of the cytotoxic potential of the isolated compounds against three cultured human tumor cell lines [4]. HL-60

13

C NMR 125 MHz)a.

2

1

No.

δC

δH mult. (J in Hz)

δC

δH mult. (J in Hz)

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 28 29 30

38.5 26.3 81.2 43.7 47.5 18.1 32.5 42.5 54.9 36.6 125.9 127.1 133.2 41.1 25.6 33.3 48.7 136.6 40.9 32.7 37.4 36.2 63.9 13.5 18.8 17.0 20.0 178.9 32.3 24.3

1.14 m, 1.87 m 2.08 m, 2.31 m 4.30 m – 1.78 m 1.41 m, 1.78 m 1.27 m, 1.43 m – 2.14 m – 6.63 dd (2.5, 10.5) 5.73 d (10.4) – – 1.03 s, 1.95 m 1.76 m, 2.23 m – – 2.14 m, 2.70 s – 1.33 m, 1.69 m 1.49 m, 2.60 m 3.91 s, 4.30 s 1.10 s 0.99 s 1.07 s 1.03 s – 0.90 s 0.88 s

38.8 26.8 88.5 39.5 56.1 18.5 33.2 39.7 48.0 37.0 23.7 122.5 145.0 42.1 28.3 23.7 46.6 42.0 46.4 30.9 34.2 33.1 28.2 17.2 15.5 17.4 26.2 180.2 33.3 23.7

0.89 m, 1.43 m 1.82 m, 2.08 m 3.21 m – 0.73 m 1.64 d (6.1) 1.22 m, 1.43 m – 1.62 m – 1.87 m 5.45 br s – – 1.20 m, 2.15 m 1.94 m, 2.12 m – 3.29 dd (3.95, 10.3) 1.28 s, 1.79 m – 1.20 m, 1.43 m 1.82 m, 2.03 m 1.30 s 1.16 s 0.80 s 0.99 s 1.30 s – 0.96 s 0.99 s

a

761

No. 1 2 3 4 5 1 2 3 4 5 6 1 2 3 4 5

2 δC Ara 104.8 75.4 75.4 69.8 66.5 Rha 101.4 72.0 81.1 72.5 70.2 18.4 Rib 104.7 72.9 68.4 69.8 65.1

1 2 3 4 5 6

Assignments aided by the DEPT, DQCOSY, HMQC, HMBC, TOCSY, and NOESY experiments.

δH mult. (J in Hz) 5.06 d (6.8) 4.02 m 4.59 t 4.12 m 3.66 d (10.9), 4.24 m 6.35 s 4.90 s 4.75 m 4.34 m 4.15 m 1.54 d (6.2) 5.98 d (4.0) 4.42 m 4.49 m 4.12 m 4.11 m, 4.34 m

δC Xyl 105.6 77.5 76.8 79.2 64.3 Rha 101.5 72.0 81.2 72.8 69.9 18.6 Rib 104.7 72.9 68.8 70.4 65.3 Glu 103.9 74.5 78.3 71.4 78.8 62.3

δH mult. (J in Hz) 4.74 d (8.8) 4.24 m 4.16 m 4.20 m 3.64 d (10.2), 4.36 m 6.45 s 4.92 s 4.73 m 4.46 m 4.72 m 1.63 d (6.1) 5.96 d (4.2) 4.32 m 4.50 m 4.17 m 4.17 m, 4.35 m 5.00 d (7.8) 3.98 m 4.19 m 4.21 m 3.97 m 4.33 m, 4.49 m

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11

13

COOH 28

OH H

3

O O

HO H CH 3 HO H

HO

H

OH

O

H OH

O

O

H CH 2OH

H

1

H OH OH H 12

COOH

OH O

HO HO H

H

H OH H

O HO

H H

HO

H

CH3 HO O O

28

1

O

3

O O H

O

H

H

H HMBC

OH

H OH OH H

2

NOESY

Fig. 2. Key HMBC and NOESY correlations of compounds 1 and 2.

(human leukemia) was cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL benzyl penicillin and 100 U/mL streptomycin in 25 cm2 culture flasks at 37 °C in humidified atmosphere with 5% CO2, Hep-G2 (human hepatocellular carcinoma) and U251MG (human glioblastoma) were cultured on DMEM medium with the same culture method of HL-60. For the cytotoxicity tests, cells in exponential growth stage were obtained from culture by trypsin digestion and centrifuging at 180 ×g for 3 min (except HL-60), then resuspended in fresh medium at a cell density of 1 × 105 of HL-60, 3 × 10 4 of Hep-G2, and 5 × 103 of U251MG per mL. The cell suspension was dispensed into a 96-well microplate at 180 μL per well, and incubated in humidified atmosphere with 5% CO2 at 37 °C for 24 h, and then treated with the compounds and positive control agents at various concentrations (2.5, 5, 10, 20, 40, 80 μM). After 72 h of treatment, 20 μL of 5 mg/mL MTT solution was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (150 μL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested with Doxorubicin (Sigma, ≥98%) as the positive control against HL-60 cells and Hep-G2 cells, and nimustine (ACNU Sigma, ≥98%) as the positive control against U251MG

cells. Dose response curves were plotted for the samples and the IC50 values were calculated as the concentrations of the test compounds resulting in 50% reduction of absorption compared with the control cells. The data represented the mean± SD of three independent experiments in which each compound concentration was tested in three replicate wells. 3. Results and discussion The 70% EtOH extract of the air-dried roots of C. argentilucida was suspended in water, and then partitioned successively with petroleum ether and n-BuOH saturated by H2O. The n-BuOH layer was dried and subjected to several chromatographic purification steps to afford 1–3. Compound 3 was deduced as cussonside B originally isolated from Cussonia barter by comparing of its physical and spectroscopic data (Supporting information) with literature values [9], which was isolated from the genus Clematis for the first time. Compound 1, a white amorphous powder with mp 235– 236 °C (dec), [α]D22 =−23.5 (c = 0.01, MeOH), was positive to Liebermann–Burchard and Molish tests. The positive ion mode HR-ESI-MS showed pseudomolecular ion peak at m/z 903.4927 [M+ Na]+ (calcd. for C46H72O16Na 903.4925), which, together

W. Hai et al. / Fitoterapia 83 (2012) 759–764

with the pseudomolecular ion peak at m/z 915 [M+Cl]− in negative ion mode ESI-MS and NMR data (Table 1), enabled the molecular formula to be determined as C46H72O16. Saponin 1 displayed 46 carbon signals in its 13C NMR spectrum, of which 30 could be assigned to the signals of the aglycone. Standard NMR analysis using a combination of 1H, 13 C and DEPT experiments quickly established that 1 was a triterpenoid sapogenin containing two double bonds. The 1H NMR spectrum contained signals for six tertiary methyl groups at δH 0.88 (3H, s, H3-30), 0.90 (3H, s, H3-29), 0.99 (3H, s, H3-25), 1.03 (3H, s, H3-27), 1.07 (3H, s, H3-26) and 1.10 (3H, s, H3-24), together with the 13C NMR signals for δC 24.3 (C-30), 32.3 (C29), 18.8 (C-25), 20.0 (C-27), 17.0 (C-26), and 13.5 (C-24), one olefinic group at δH 6.63 (1H, dd, J = 2.5, 10.5 Hz, H-11), 5.73 (1H, d, J = 10.4 Hz, H-12) and δC 125.9 (C-11), 127.1 (C12), another olefinic group at δC 133.2 (C-13), 136.6 (C-18), a hydroxymethyl group at δH 3.91, 4.30 and δC 63.9 (C-23), and a carboxy group at δC 178.9 (C-28), which were characteristic for the oleanane skeleton with a carboxy group at C-17, two double bonds located at the Δ 11(12), and Δ13(18) position and a hydroxyl group at C-23. The assignments of the NMR signals associated with the aglycone moiety were derived from 1 H–1H COSY, TOCSY, HSQC, HMBC and NOESY experiments. These data revealed the common hederagenin aglycone for 1 [10,11]. The two double bonds could only be located at the Δ 11(12), and Δ 13(18) position, which was identified by the cross-peaks in the HMBC spectrum (Fig. 2). The observation of HMBC correlations from H-9 δH 2.14 (m) to C-11 and C-12 allowed us to determine one of the double bonds located at C-11. Similarly, the observation of HMBC correlations from H2-19 (δH 2.14, 2.70) to the olefinic quaternary carbon C-13, as well as H3-27, H2-19 and H-12 to C-18 indicated the other double bond located at C-13. The C-3 carbon was observed at δC 81.2 and the C-28 carbonyl carbon at δC 178.9, which suggested that no sugar linkage was formed at C-28 and that an oligosaccharide moiety was attached at C-3 [12]. The observation of correlations of H-3 with H-23α and H-5 in the NOESY spectrum indicated the βconfiguration for the 3-O-sugar moiety (Fig. 2). The sugar moieties of 1 were determined to be L-arabinose, L-rhamnose, and D-ribose in a ratio of 1:1:1 by acidic hydrolysis followed by comparing the GC analysis of the corresponding aldononitrile peracetates with those of the authentic samples prepared in the same manner in the literature [13]. The 1H NMR spectrum of 1 displayed three signals ascribable to the anomeric protons (δH 5.06, 5.98, and 6.35), and one methyl group of 6-deoxyhexopyranosyl moiety at δH 1.54 (3H, Rha H3-6), which were correlated in the HSQC experiment with carbon signals at δC 104.8 (3-Ara C-1), 104.7 (Rib C-1), 101.4 (Rha C-1), and 18.4 (Rha C-6), respectively. The α anomeric configurations for arabinose and β anomeric configurations for ribose units were determined from their 3JH-1/H-2 coupling constants (J = 6.8 Hz and 4.0 Hz). Although the anomeric proton of the rhamnose unit was observed as a singlet in the 1H NMR spectrum, the 13C -NMR shift of Rha C-5 at δC 70.2 indicated the α anomeric configuration [11]. All proton signals due to sugars were identified by careful analysis of the 1H–1H COSY, TOCSY and NOESY spectra, and the carbon signals were assigned by HSQC and further confirmed by HMBC spectrum. Data from the above experiments (Table 1) indicated that three sugar residues were in the pyranose forms. A thorough inspection

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of HMBC and NOESY spectra led to the determination of conjunction of sugar chain. In the HMBC spectrum, a cross peak between H-1 of arabinose (Ara) and C-3 of the aglycone indicated that Ara was connected to C-3 of the aglycone. The linkage of the terminal Rhamnose (Rha) at C-2 of Ara was indicated by the cross peak Rha H-1/Ara C-2. Similarly, the linkage of the terminal ribose (Rib) at C-3 of Rha was indicated by cross peaks Rib H-1/Rha C3. The conclusion was confirmed by NOESY correlations as shown in Fig. 2. On the basis of the above evidences, the structure of compound 1 was determined as 3β-O-[β-D-ribopyranosyl(1→ 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl] hederagenin-11,13-dien-28-oic acid. Compound 1 is characterized at the two double bonds in the aglycone of C-11 and C-13, which is a very rare structure feature among naturally occurring triterpenoid saponins, and it's the first time that this aglycone has been found in the genus Clematis. Compound 2, a white amorphous powder with mp 244– 245 °C, [α]D22 = −21.1 (c = 0.09, MeOH), was positive to Liebermann–Burchard and Molish tests. The molecular formula was established as C52H84O20 from the [M + Na] + ion at m/z 1051.5461 (calcd. for C52H84O20Na 1051.5454) in the positive ion mode HR-ESI-MS. The 1H NMR and 13C NMR spectra of 2 showed seven tertiary methy groups at δH 0.80 (3H, s, H3-25), 0.96 (3H, s, H3-29), 0.99 (3H, s, H3-30), 0.99 (3H, s, H3-26), 1.16 (3H, s, H3-24), 1.30 (3H, s, H3-23), and 1.30 (3H, s, H3-27) and one trisubstituted olefinic proton at δH 5.45 (1H ,br s, H-12) coupled with seven sp 3 carbons at δC 15.5 (C-25), 33.3 (C-29), 17.4 (C-26), 23.7 (C-30), 17.2 (C24), 28.2 (C-23), and 26.2 (C-27) and two sp2 olefinic carbons at δc 122.5 (C-12) and 145.0 (C-13) (Table 1). The aglycone of 2 was identified as oleanolic acid by extensive 2D NMR studies [12]. The chemical shifts C-3 (δC 88.5) and C-28 δC (180.2) revealed that 2 was a monodesmosidic glycoside, too. The presence of D-xylose, L-rhamnose, D-ribose, and D-glucose in a 1:1:1:1 ratio was established by acid hydrolysis followed by GC analysis of the corresponding aldononitrile peracetates. The 1H NMR spectrum of 2 exhibited four sugar anomeric protons at δH 4.74 (1H, d, J = 8.8 Hz, 3-Xyl H-1), 6.45 (1H, br. s, Rha H-1), 5.96 (1H, d, J = 4.2 Hz, Rib H-1), and 5.00 (1H, d, J = 7.8 Hz, Glc H-1) which were correlated with carbon signals in the HSQC experiment at δC 105.6 (3-Xyl C-1), 101.5 (Rha C1), 104.7 (Rib C-1), and 103.9 (Glc C-1), respectively. From the coupling constants of the anomeric signals, the above four sugars were deduced to be β-configuration for D-xylose, D-glucose and D-ribose, and α-configuration for L-rhamnose. Complete 1H and 13C NMR assignments of the oligosaccharide part were achieved by a combination of 2D NMR experiments (Table 1). The sequence and binding sites of the oligosaccharide chain connected to C-3 of the aglycone were deduced mainly by the HMBC experiment. In the HMBC spectrum of 2, longrange correlations between the anomeric protons at H-1 of Xyl with C-3, H-1 of Glc with C-4 (δC 79.2) of Xyl, H-1 of Rha with C-2 (δC 77.5 ) of Xyl, and H-1 of Rib with C-3 (δC 81.2) of Rha indicated that the oligosaccharide chain of C-3 was 3-O{β-D-ribopyranosyl-(1→ 3)-α-L-rhamnopyranosyl-(1 → 2)-[βD-glucopyranosyl-(1 → 4)]-β-D-xylopyranosyl}. The result was confirmed by NOESY spectrum (Fig. 2). Hence, compound 2 was established as 3β-O-{β-D-ribopyranosyl-(1→ 3)-α-Lrhamnopyranosyl-(1→ 2)-[β-D-glucopyranosyl -(1→ 4)]-β-Dxylopyranosyl} oleanolic acid.

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Table 2 Cytotoxicity of glycosides 1–3 against three cancer cell lines in vitro (IC50, μM).a Cell line

1

2

3

Positive control

HL-60 Hep-G2 U251MG

6.61 ± 1.40 5.30 ± 1.90 25.40 ± 0.87

3.82 ± 0.28 2.74 ± 0.34 10.35 ± 1.34

>100 >100 >100

0.35 ± 0.03b 0.52 ± 0.05b 0.92 ± 0.04c

a IC50 values are means from three independent experiments (average ± SD) in which each compound concentration was tested in three replicate wells. b Doxorubicin as positive control. c Nimustine (ACNU) as positive control.

Institute for the measurement of NMR spectra, and Prof. Guang Cheng, Department of Neurosurgery of Xijing Hospital, Fourth Military Medical University for the measurement of cytotoxicity. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.fitote.2012.03.003. References

The cytotoxicities of saponins 1–3 against human leukemia HL-60 cell lines, human hepatocellular carcinoma Hep-G2 cell lines and human glioblastoma U251MG cell lines were determined using the MTT colorimetric assay. The results of the cytotoxic activities of these saponins against human cancer cell lines in vitro were shown in Table 2. Among the tested compounds, 1 and 2 possessed a free carboxylic group at C-28 exhibiting significant cytotoxicity against all of the test cancer cell lines, earlier studies on the cytotoxicity of similar compounds against HL-60 and U251MG cell lines have reached the same result [14,15]. However 3 was a monodesmoside without the free carboxylic group at C-28 and no cytotoxic activity was observed which differed from the reported result of another literature in terms of the cytotoxicity against HL-60 cell lines [16]. Hence, a more comprehensive cytotoxicity study covering more potential impact factors, like cell culturing environment, district differentiation of HL-60 cell lines etc., is necessary to understand the difference. As two new saponins (1 and 2) possessed different oleanolic acid aglycones and oligosaccharide chains, the fact of saponin 2 exhibiting more activity than 1 in the cytotoxic experiment suggested that the cytotoxicity of these class of saponins were related to their aglycones and oligosaccharide chains, and further studies on the cytotoxicity of the eleven saponins previously isolated from C. argentilucida are necessary to clarify their clear structure– activity relationship. Acknowledgments The authors are grateful to Mr. Minchang Wang, Nuclear Magnetic Resonance Center, Xi'an Modern Chemistry Research

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