Novel Steroidal Glycosides from the Bulbs of Lilium pumilum

Examination of the bulbs of Lilium pumilum (Liliaceae) led to the isolation of four novel steroidal glycosides (1–4) with a 2,3,4-trisubstituted β-d-glucopyranosyl unit. In 1 and 3, the α-l-arabinopyranosyl moiety is linked to C-3 of the inner trisubstituted β-d-glucopyranosyl group and is present as an usual 4C1 conformation. In contrast, in 2 and 4, the α-l-arabinopyranosyl moiety, which is attached to C-4 of the inner trisubstituted β-d-glucopyranosyl group, is present as a 1C4 conformation. The structures of the new steroidal glycosides were determined based on the results of spectroscopic analyses, including two-dimensional (2D) NMR data and hydrolysis.


Introduction
Lilium pumilum D.C. (Liliaceae) is described in the Japanese Pharmacopoeia (16th edition) as a plant from which the crude drug Lilium Bulb is derived.Lilium Bulb has long been used as an antitussive and anti-inflammatory agent in traditional Chinese medicine [1].The bulbs of L. pumilum (L.tenuifolium Fisch.ex Hook.) contain phenylpropanoid derivatives, such as regalosides A and D, and sterol glycosides, such as tenuifoliosides A and B [2].Although plants belonging to the family Liliaceae are rich sources of bioactive steroidal glycosides, such as OSW-1 or galtonioside A with cytotoxic activities against tumor cells [3,4], few studies have focused on steroidal glycosides of L. pumilum [5].Our preliminary analysis

OPEN ACCESS
of the MeOH extract of L. pumilum bulbs suggests that it contains more steroidal glycosides.Here, we report four novel steroidal glycosides (1-4) with a trisubstituted β-D-glucopyranosyl unit isolated from the bulbs of L. pumilum.The structures of the new steroidal glycosides were determined based on the results of spectroscopic analysis, including two-dimensional NMR data, and hydrolysis followed by chromatographic and spectroscopic analyses.

Results and Discussion
The bulbs of L. pumilum (1.4 kg fr.wt) were extracted with MeOH.The MeOH extract (65 g) was passed through a porous-polymer polystyrene resin (Diaion HP-20) column, and the MeOH-eluted fraction (1.8 g) was subjected to silica gel and octadecylsilanized (ODS) silica gel column chromatography (CC), giving 1-4 (Figure 1).which were characteristic of 22-hydroxyfurostanol glycosides [6].Acid hydrolysis of 1 with 1 M HCl yielded 1a as the aglycone and arabinose, glucose and rhamnose as the carbohydrate moieties.The monosaccharides and their absolute configurations were identified by direct HPLC analysis of the hydrolysate; L-arabinose, D-glucose and L-rhamnose, respectively.The aglycone (1a) was identified as (25R)-spirost-5-en-3β-ol (diosgenin) from its physical and spectroscopic data [7].These NMR data, the chemical evidence, and the positive color reaction with Ehrlich's reagent suggested that 1 was a 22-hydroxyprotodiosgenin bisdesmoside, the sugar moieties of which consisted of five monosaccharides.The 1 H-1 H COSY and 1D TOCSY spectra of 1 allowed the 1 H-NMR chemical shifts, signal multiplet patterns, and coupling constants of the sugar moieties to be assigned as shown in Table 2.The 1 H-NMR signals were associated with the corresponding one-bond coupled carbons using the HMQC and HSQC-TOCSY spectra, leading to the assignments of all the 13 C-NMR chemical shifts of the sugar moieties.Comparing the 13 C-NMR chemical shifts of each monosaccharide and reference methyl glycosides indicated the presence of a substituted β-D-glucopyranosyl ( 4 C1) unit (Glc (I)), two β-D-glucopyranosyl ( 4 C1) units (Glc (II) and Glc (III)), an α-L-arabinopyranosyl ( 4 C1) unit (Ara), and an α-L-rhamnopyranosyl ( 1 C4) unit (Rha) as the terminal glycosyl groups [8].The 13 C-NMR shifts of the inner Glc (I) moiety (δC 99.8, 78.9, 79.8, 72.5, 78.5, and 60.9) suggested that its C-2, C-3, and C-4 hydroxy groups were substituted with the other sugar moieties.The anomeric configurations of the Ara and Glc groups were ascertained as α and β, respectively, from the relatively large J values of their anomeric protons (5.4-7.8Hz) [7].For the Rha group, the 13 C-NMR chemical shifts (δC 102.4,72.4,72.8, 73.8, 70.0, and 18.6) suggested an α-pyranoid anomeric form.The HMBC correlations between the anomeric proton (H-1) of Ara at δH 5.56 and C-3′ of Glc (I) at δC 79.8, between H-1′′ of Glc (II) at δH 5.39 and C-4′ of Glc (I) at δC 72.5, between H-1 of Rha at δH 6.07 and C-2′ of Glc (I) at δC 78.9, and between H-1′ of Glc (I) at δH 4.92 and C-3 of the aglycone at δC 77.9
Compound 3 was analyzed as C56H90O26 based on the HR-ESI-TOF-MS (m/z 1201.5615[M + Na] + ) and 13 C-NMR (56 carbon signals) data.This molecular formula was smaller than that of 1 by 18.0059 (H2O).The 1 H-and 13 C-NMR spectral features of 3 were similar to those of 1; however, the Me-21 doublet signal at δH 1.34 (d, J = 6.9 Hz) in the 1 H-NMR spectrum of 1 was replaced by a methyl singlet signal at δH 1.64 in that of 3, and an olefinic functionality (δC 152.3 and 103.6) was observed in addition to the 5(6)-ene group in the 13 C-NMR spectrum of 3.These spectroscopic data and the HMBC correlations from H-17 at δH 2.45, H2-23 at δH 2.22, and Me-21 at δH 1.64, to C-22 at δC 152.3 and C-20 at δC 103.6 indicated that 3 was the corresponding Δ 20(22) -pseudo-furostanol glycoside of 1.This was supported by all other spectroscopic data and the results of acid hydrolysis.The structure of 3 was assigned as (25R)-26-[(β-Dglucopyranosyl)oxy]-furosta-5,20

Plant Material
The L. pumilum bulbs were obtained from the Sakata Seed Corporation (Yokohama, Japan) in 2008 and were identified by Dr. Yutaka Sashida, professor emeritus of Tokyo University of Pharmacy and Life Sciences.We have retained a voucher specimen in our laboratory (KS-2008-002).

Cell Culture Assay
Cell growth was measured with an MTT reduction assay, as described in a previous paper [12].

Conclusions
In conclusion, four new steroidal glycosides (1-4) were isolated from the bulbs of L. pumilum, and they were classified into furostanol glycosides (1 and 2) and Δ 20(22) -pseudo-furostanol glycosides (3 and 4).Compounds 1-4 are novel steroidal glycosides with a 2,3,4-trisubstituted β-D-glucopyranosyl moiety at C-3 of the aglycone.In general, α-L-arabinopyranosyl groups are stable in a 4 C1 conformation in glycosides, except for those glycosylated at C-2 [13].In 1 and 3, the α-L-arabinopyranosyl moiety is linked to C-3 of the inner trisubstituted β-D-glucopyranosyl group and is present as an usual 4 C1 conformation.In contrast, in 2 and 4, the α-L-arabinopyranosyl moiety, which is attached to C-4 of the inner trisubstituted β-D-glucopyranosyl group, is present as a 1 C4 conformation.It is notable that the 1 C4 α-L-arabinopyranosyl moiety in 4 was converted to the 4 C1 moiety on peracetylation (4a).These interesting steric behaviors of arabinopyranose cannot be explained by steric hindrance only.In a recent study, there

Compound 2
had the same molecular formula as 1 of C56H92O27, based on the HR-ESI-TOF-MS and 13 C-NMR (56 carbon signals) data.Two singlet signals for tertiary methyl groups at δH 1.04 and 0.89, two doublet signals for secondary methyl groups at δH 1.33 (d, J = 6.9 Hz) and 1.00 (d, J = 6.8 Hz), and five signals for anomeric protons at δH 5.94 (br s), 5.74 (d, J = 1.2 Hz), 5.49 (d, J = 7.8 Hz), 4.85 (d, J = 8.0 Hz), and 4.81 (d, J = 7.8 Hz) were observed in the 1 H-NMR spectrum of 2. The 13 C-NMR spectrum contained a signal for an acetal carbon at δC 110.6, and signals for four steroid methyl groups at δC 19.3, 17.4, 16.4, and 16.4.These spectroscopic data for 2 were analogous to those of 1, and suggested that 2 shared the same fundamental furostanol skeleton as 1.Acid hydrolysis of 2 gave 1a, L-arabinose, D-glucose, and L-rhamnose.