Cytotoxic Steroidal Saponins from the Flowers of Allium leucanthum

Allium leucanthum C. Koch is an endemic Caucasian species that grows in Georgia. The flowers are used in traditional medicine. Phytochemical investigation allowed the isolation of seven spirostanol type saponins from the flowers. Their structures were elucidated on the base of NMR and HRESIMS spectrometry data. A new compound, which we have named leucospiroside A (5), has been identified as (25R)-5α-spirostane-2α,3β,6β-triol 3-O-β-glucopyranosyl-(1→3)-β-glucopyranosyl-(1→2)-[β-glucopyranosyl-(1→3)]-β-glucopyranosyl-(1→4)-β-galactopyranoside. The six others were known substances, but are described in this plant for the first time. The crude extract, spirostanol and furostanol fractions, as well as isolated compounds, were evaluated for their in vitro cytotoxic activity. Compounds 1-3 and 5 were found to be the most active, with relatively similar IC50 values ranging from 3.7 to 5.8 µM for a lung cancer cell line (A549) and 5.6 to 8.2 µM for a colon cancer cell line (DLD-1).


Introduction
Genus Allium includes up to 500 species in the world flora. Among them, some 70 grow in the Caucasian region and 35 species are described in Georgia [1]. Allium leucanthum C. Koch (Alliaceae), called "whiteflower onion" in Georgia, is a Caucasian endemic species [2] and, along with other Allium species, is widely used in Georgian traditional medicine as an antifungal, antiseptic and antibacterial remedy [3][4]. Various secondary metabolites were identified in genus Allium [5]. Among them, steroidal saponins have been investigated for their antibacterial [6], antifungal [7][8] and antioxidant [9][10] activities. Furthermore, steroidal saponins isolated from different species of onions showed a significant cytotoxic activity against murine fibrosarcoma [11], lung carcinoma [12], human melanoma [13] and human leukemia [14].
Steroidal saponins of only one species, A. erubescens, which grows in Georgia, have been reported [15]. This prompted us to undertake the phytochemical and cytotoxic studies of different extracts prepared from Allium leucanthum flowers. This bioassay-guided isolation allowed us to obtain seven cytotoxic spirostanol saponins (1-7, Figure 1).  This paper describes the isolation of several spirostanol saponins from the flowers of Allium leucanthum C. Koch, and the structural determination of new compound 5 by HRESIMS, 1 H and 13 C NMR spectroscopy. Furthermore, the in vitro cytotoxic activities of saponins and related aglycons are reported.

Results and Discussion
The flowers of Allium leucanthum C. Koch were extracted with aqueous methanol. The cytotoxicity of the extract was evaluated against human tumor and healthy cell lines including lung adenocarcinoma (A549), colorectal adenocarcinoma (DLD-1) and normal skin fibroblasts (WS1) ( Table 1). The methanolic extract was shown to strongly inhibit the growth of tumor cell lines, with an IC 50 of 15 ± 3 µg/mL for A549 and 19.6 ± 0.9 µg/mL for DLD-1. However, the extract was not found to be selective toward tumor cell lines when compared with healthy cell line WS1 (IC 50 , 10.6 ± 0.8 µg/mL). The methanolic extract was then partitioned between water and 1-butanol to eliminate hydrophylic compounds. The 1-butanol extract was then chromatographied on Diaion resin under gradient conditions with MeOH-H 2 O (0→100%). Two fractions were obtained and were shown to contain spirostanol and furostanol saponins. The cytotoxicity of both fractions was tested against tumor and normal cell lines. The spirostanol fraction was more active than the methanolic extract. In contrast, the furostanol fraction was inactive against both tumor cell lines (IC 50 > 90 µg/mL). Therefore, the chemical composition of the spirostanol fraction was studied and seven compounds 1-7 were isolated using open column chromatography. The structures of the known compounds (1-4 and 7) were determined by comparison of the 1 H-and 13 C-NMR spectral data with those reported in the literature. They were identified for the first time in Allium leucanthum as yayoisaponin C (1) [16], eruboside B (2) [7,15], aginoside (3) [17,18] [20]. Compound 6, namely (25R)-5α-spirostane-2α,3β,6β-triol 3-Oβ-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, was already reported in the literature [17], but since NMR data were missing, they are reported in this paper (Table 3). . The 13 C NMR spectrum showed 57 peaks: 30 for the sugar moieties including five anomeric carbons at δ C 105.0, 104.0, 103.9, 103.5 and 102.7, and 27 for the aglycones part. The latter were found very similar to those of 1, suggesting that the aglycone of 5 was also agigenin. The NMR chemical shifts for galactose and glucose A-C, as determined from HSQC and multiple 1D-selective TOCSY experiments, were very similar to those reported for 1 whereas C-3 of glucose B of 5 (δ C 87.6) was significantly shifted downfield by 9.8 ppm from that of 1 (δ C 77.8), indicating that another sugar unit was attached to C-3 of glucose B. The same sugar units were also identical to those of yayoisaponin A [16] except for xylose which was identified as glucose (glucose D) in compound 5 based on the 13 C-NMR chemical shift [21]. Indeed, the identification of the sugar units was confirmed by acid hydrolysis of compound 5 where only glucose and galactose could be detected by TLC analysis. These sugar linkages were confirmed by long range correlations on the HMBC spectrum between H-1 of Gal (δ H 5.00) and C-3 of aglycone (δ C 84.  Table 4 show that compounds 1-3 and 5 possess a relatively similar cytotoxicity against both tumor cell lines, with IC 50 values ranging from 3.7 to 5.8 µM for A549 and 5.6 to 8.2 µM for DLD-1. The cytotoxic activities of compounds 1-3 were previously reported in the literature [16,22]. When compared with their cytotoxic activities against healthy cell line WS1, the glycosides were not found to be selectively toxic towards the cancer cell lines. The structure-activity relationship shows that the presence of glucose or xylose at position R 2 of compounds 1 and 3 is important for bioactivity. Indeed, the cytotoxicities of compound 1 and 3 are significantly (p<0.05, ANOVA one way analysis) increased by about three to six times when compared with compound 6 which has neither glucose or xylose at position R 2 . However, the addition of a second glucose at position R 3 (compound 5) does not significantly improve cytotoxic activity when compared with compound 1 (p<0.05). The change from a glucose (compound 1) to a xylose at position R 2 (compound 3) with a hydroxyl at position R 1 does not modify the cytotoxicity of the molecule. However, the absence of a hydroxyl at position R 1 slightly reduces the activity.   In conclusion, the steroidal saponin leucospiroside A (5) reported for the first time, was isolated from Allium leucanthum along with six other saponins (1-4 and 6-7) which were described for the first time in this endemic species. Compounds 1-3 and 5 were found to be the most active compounds. For all steroidal saponins, the presence of sugar moieties is important for the activity.

Experimental Section
General experimental IR spectra were recorded (neat) using a Perkin-Elmer Spectrum One spectrometer. 1 H-and 13 C-NMR chemical shifts in ppm were referenced with the residual solvent (CD 3 OD) signals (δ H 3.31 and δ C 49.0) or with TMS as internal standard (for pyridine-d 5

Acid hydrolysis of leucospiroside A (5)
A solution of compound 5 (2 mg) in HCl 5% (3 mL) was heated at 100 °C for 4 h. The reaction mixture was treated with chloroform and the aq. phase was neutralized with N,N-dioctylmethylamine (10% in CHCl 3 ) and dried. The monosaccharide part was dissolved in MeOH 50% (1 mL), and analyzed by TLC with reference galactose (R f = 0.33) and glucose (R f = 0.35). TLC identification of monosaccharides was carried out in a CH 2 Cl 2 -CH 3 OH-H 2 O (50:25:5) solvent system, and further developed using an orthophosphoric acid soln. of naphtorezorsinol 5% in EtOH, followed by heating at 110 °C.