Steroidal and Phenolic Glycosides from the Bulbs of Lilium pumilum DC and Their Potential Na+/K+ ATPase Inhibitory Activity

A new steroidal saponin, named pumilum A (1), and a new phenolic glycoside, threo-1-(4′-hydroxy-2′-methoxyphenyl)-2-(2′′,4′′-dihydroxyphenyl)-1,3-propanediol-4′-O-β-D-glucopyranoside (7) were isolated from the methanolic extract of the bulbs of Lilium pumilum DC, along with five known steroidal saponins. Their chemical structures were elucidated on the basis of detailed spectroscopic analysis, including 1D and 2D NMR techniques and chemical methods. In addition, the inhibitory activity of all the isolates on Na+/K+ ATPase was evaluated.


Introduction
"Bǎi-hé", a Chinese crude drug, is prepared from the bulbs of the Lilium species and is regularly used nowadays in China as a sedative, antitussive and anti-inflammatory agent [1][2][3][4]. Previously, OPEN ACCESS phenolic glycosides, steroidal saponins and steroidal alkaloids have been isolated from the bulbs of the Lilium species [5][6][7][8][9][10][11][12]. However, to date, a survey of the literature showed that no chemical work has been done on Lilium pumilum DC.
Our studies indicated that the methanolic extract of the bulbs of Lilium pumilum DC showed significant Na + /K + ATPase inhibition activity (IC 50 value: 0.5 × 10 −5 M). To further investigate the constituents and screen the bioactive constituents from its bulbs, a phytochemical study was performed that resulted in the isolation of new compounds 1 and 7, along with five known steroidal saponins. In the present article, we describe the structural elucidation of new compounds 1 and 7, together with the Na + /K + ATPase inhibitory activity of all these compounds 1-7 ( Figure 1).

Results and Discussion
The methanolic extract from the bulbs of Lilium pumilum DC was successively subjected to column chromatography over silica gel, ODS, Diaion HP-20 and semipreparative HPLC to afford six steroidal saponins and a new phenolic glycoside. All compounds were obtained from this plant for the first time.
Compound 1 had the molecular formula C 39 H 62 O 15 , as deduced from the positive-ion HR-ESI-MS (m/z 793.6704 [M+Na] + ) and 13 C-NMR spectrum. The glycosidic nature of 1 was indicated by the strong absorption bands at 3435 cm −1 and 1045 cm −1 in the IR spectrum. The typical absorptions at 975, 915, 895, and 860 cm −1 in the IR spectrum suggested that 1 was a spirostanol saponin [13,14]. The intensity of the absorptions (895 > 915) indicated that the absolute configuration of C-25 was R. The 1 H-NMR spectrum contained signals for two oxygenated methylene proton signals at  Table 1). The 13 C-NMR and DEPT spectrum showed a quaternary carbon signal at δ 109.7, which is the characteristic C-22 of a spirostanol skeleton [15,16]. The presence of a carbonyl group in 1 was established from the IR (1707 cm −1 ) and 13 C-NMR spectra (δ 209.4). The existence of D-glucose and L-rhamnose were determined by hydrolysis of 1 with 1 M HCl and GC analysis. The J values of the anomeric proton signals indicated the β-and α-configuration at the anomeric centers of D-glucose and L-rhamnose, respectively. These findings showed that 1 was a (25R)-spirostanol diglycoside.
The inhibitory activity of 1-7 on Na + /K + ATPase was assayed. Ouabain was used as a positive control (IC 50 value: 0.1 × 10 −5 M). Saponins 3 and 4 were found to inhibit sensitive Na + /K + ATPase with the IC 50 values of 7.3 × 10 −5 and 9.1 × 10 −6 M, respectively, while the others were inactive [IC 50 > 1.0 × 10 −4 M]. Thus, this research suggested that the saponins from the methanolic extract may be primary Na + /K + ATPase inhibitors. The weak Na + /K + ATPase inhibitory activity of the initial extract (compounds 1-7) is thus presumably due to other as yet unidentified compounds. One possible reason for this is that compounds with highly Na + /K + ATPase inhibitory activity are low in the BuOHsoluble fraction of the methanolic extract from the bulbs of Lilium pumilum DC. Another possible reason is compounds with increased activity may be found in other extraction fractions (ethyl acetate extract, chloroform extract and petroleum ether extract) of the methanolic extract from the bulbs of this medicine plant. Therefore, more chemical work needs to be done in the future.

General
Specific rotation measurements were recorded on a Perkin-Elmer 242 MC polarimeter. UV spectra were recorded on a Hewlett-Packard HP-845 UV-VIS spectrophotometer. IR spectra were recorded on a Nicolet 470 spectrometer and MS on a Varian MAT-212 mass spectrometer and a Shimadzu GC-MS model QP2010 Plus spectrophotometer, respectively. NMR spectra were recorded on a Bruker AM-400 spectrameter (400 MHz for 1 H-NMR) or a Bruker DRX-500 (500 MHz for 1 H-NMR) spectrameter using standard Bruker pulse programs. Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as internal standard. Column chromatography separations were carried out on silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd, Qingdao, China), ODS (50 mesh, AA12S50, YMC), Diaion HP-20 (Pharmacia, Peapack, NJ, USA) and Sephadex LH-20 (Pharmacia, Peapack, NJ, USA). Ouabain sensitive dog kidney Na + /K + ATPase was obtained from Sigma (Shanghai, China). All other chemicals used were of biochemical reagent grade.

Plant Material
The bulbs of Lilium pumilum DC were collected in Lanzhou, Gansu Province of China in September 2010, and were identified by one of the authors (Chun-Yan Fu of Department of Pharmacy, Shaoyang Medical College Level Speciaity School, Shaoyang). A voucher specimen (NO. 20100908) has been deposited in the authors' laboratory.

Extraction and Isolation
Fresh bulbs of Lilium pumilum DC (5 kg) were cut into pieces and extracted with methanol (3 × 8 L, 3h each) under reflux. The solvent was removed at reduced pressure to give a viscous residue (328 g). The entire crude extract was suspended in H 2 O (3 L), and extracted with n-BuOH five times (3 L each) to give an n-BuOH extract (206 g). This extract was then fractionated by silica gel column chromatography (column: 90 cm × 9 cm) using a mobile phase composed of CHCl 3 Table 2.

Acid Hydrolysis of Compound 1
A solution (2.5 mg) of 1 in 1 M HCl (1 mL) was heated at 100 °C for 2 h under an N 2 atmosphere. After cooling, the solution was removed by blowing with N 2 . The residue was dissolved in a solution of 1-(trimethylsilyl) imidazole (0.5 mg) in pyridine (1.0 mL), and stirred at 60 °C for 5 min. After removal of the solvent with a stream of N 2 , the residue was partitioned between H 2 O and CH 2 Cl 2 (1:1 v/v). The CH 2 Cl 2 fraction was analyzed by GC using a L-Chirasil-Val column (0.32 mm × 25 m). The temperature of the injector and detector were 200 °C. A temperature gradient system was used for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min. The peaks of the hydrolysates of 1 were confirmed by comparison of retention times of authentic samples D-glucose, L-rhamnose treated with 1-(trimethylsilyl) imidazole [24].