Two New Steroidal Saponins from Allium macrostemon Bunge and Their Cytotoxity on Different Cancer Cell Lines

Two new steroidal saponins (1 and 2) were isolated from the dried bulbs of Allium macrostemon Bunge. Their structures were elucidated by the spectral data as 26-O-β-D-glucopyranosyl-5α-furost-25 (27)-ene-3β, 12β, 22, 26-tetraol-3-O-β-D-glucopyranosyl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside (1) and 26-O-β-D-glucopyranosyl-5β-furost-20 (22)-25 (27)-dien-3β, 12β, 26-triol-3-O-β-D-glucopyranosyl (1→2)-β-D-galactopyranoside (2), respectively. Their cytotoxic activities on several cancer cell lines (MCF-7, NCI-H460, SF-268 and HepG2) were tested. 1 showed special cytotoxity on SF-268, while 2 showed cytotoxity on NCI-H460 and SF-268 cell lines, respectively.


Introduction
The dried bulbs of Allium macrostemon Bunge are well known as the traditional Chinese medicine "Xie bai", which is used for treatment of throracic pain, stenocardia, heart asthma and diarrhea [1]. The main constituents of the ethanol extract of Allium macrostemon Bunge are steroidal saponins, which showed inhibitory effects on platelet aggregation [2] and degradation effects on blood serum OPEN ACCESS peroxides proton [3], as well as the cytotoxic activity on different cancer cell lines [4,5]. This paper describes the isolation and structural elucidation of two new steroid saponins from "Xie bai" ethanol extract. Their cytotoxic activities against several human cancer cell lines, including MCF-7 (breast), NCI-H460 (lung), SF-268 (CNS) and HepG2 (liver), were also tested.

Results and Discussion
The dried bulbs of Allium Macrotemon Bunge were extracted with 60% ethanol. The concentrated ethanol extract was passed through a Diaion HP-20 column eluting with a EtOH-H 2 O gradient. The 60% ethanol eluting fraction was collected and further fractionated by silica gel and octadecylsilanized silica gel and repeated Prep-HPLC to yield compounds 1 and 2.
Compound 1 was obtained as an amorphous powder. Comparison of the 13 C-NMR data of the aglycone of 1 with those for Macrostemonoside E suggested differences at C-12, C-20, C-22, C-25 and C-27. The absence of characteristic C 20 -C 22 double bond carbon at δ 152.4 and 103.7 and the presence of carbon signal at δ 110.9 revealed the presence of a hydroxyl group at C-22. In HMBC spectrum, the long-range correlations from δ 1.36 (H-18) to δ 79.3 (C-12) suggested the presence of hydroxyl at C-12 and correlations from olefinic proton signals at δ5.34, 5.70 (H-27) to δ 72.0 (C-26), 147.2 (C-25), 28.4 (C-24) suggested the presence of a C 25 -C 27 double bond in the aglycone. In the NOESY spectrum, the absence of NOE correlation between proton signals at d 1.36 (H-18) and 3.58 (H-12) suggested the β-orientation of the C-12 hydroxyl and this was also confirmed by the comparison of C-12 carbon signal at δ 79.3 with Macrostemonoside G [7] which has the β-orientation of the C-12 hydroxyl at δ 79.6.
With the same NMR data of the sugar parts compared with Macrostemonoside E, the sugar moiety was finally unambiguously determined by acid hydrolysis and analysis of a combination of DEPT, 1 H-1 H COSY, HMQC, HMBC and TOCSY spectra. Then, the structure of 1 was determined as 6-O-β-D- Compound 2 was also isolated as an amorphous powder. Its molecular formula of C 45 H 72 O 19 was deduced by positive-ion HR-ESIMS at m/z 939.4572 [M+Na] + (calcd. 939.4566). The 1 H-NMR spectrum contained three steroid methyl groups at δ 0.92 (3H, s), 0.99 (3H, s) and 2.00 (3H, s). These three single peak methyl proton signals, especially the signal shift downfield to δ 2.00 compared with that of signal at H-21 of 1, revealed the presence of double bonds at C 25 -C 27 and C 20 -C 22 . Comparison of the 13 C-NMR spectrum of 2 with that of Macrostemonoside G [7] showed considerable structural similarity. However, the molecular formula of 2 was lower by 18 units (one H 2 O) than that of Macrostemonoside G and the difference were recognized in the carbon signals from the ring E portion. The 13 C-NMR spectrum showed that the carbon signals of C-16, C-17, C-20, C-22 and C-24 of 2 were shifted downfield by approximately + 3.3, + 0.9, + 63.2, + 31.1 and + 2.8 ppm, respectively, while the carbon signal of C-21 and C-23 shifted to higher field by -3.3 and -13.1 ppm comparing with those of Macrostemonoside G, which suggested the presence of a double bond between C-20 and C-22. This was also confirmed by long-range correlations between the proton signal at δ 0.92 (H-21) and carbon signals at δ 104.8 (C-20) and 151.7 (C-22) in the HMBC spectrum. The HMBC correlation between the proton signal at δ 0.92 (CH 3 -18) and the carbon signal at δ 78.7 (C-12) indicated a hydroxyl group at C-12. In the NOESY spectrum, the β-orientation of the C-12 hydroxyl group was inferred due to the absence of NOE correlation between the proton signals at δ 0.92 (H-18) and 3.53 (H-12). The triglycoside moiety of 2 was shown to be the same as that Macrostemonoside G and the structure of 2 was assigned as 26 The in vitro cytotoxicity of compounds 1 and 2 against various cancer cell lines was evaluated by MTT assay. IC 50 values were calculated by the LOGIT method (Table 1). Compound 1 showed mild cytotoxity, especially towards the SF-268 cell line with IC 50 values of 35.2 μM and compound 2 showed mild cytotoxity, especially to the SF-268 and NCI-H460 cell lines, with IC 50 values of 25.7 and 35.4 μM, respectively. Comparison of the structure and the cytotoxic activity of 1 and 2 with those steroidal saponins reported previously [5] suggests that the presence of a C 25 -C 27 double bond and C-12 hydroxyl in the steroidal pentaglycoside aglycone contribute to the cytotoxicity in the SF-268 cell line and a C 20 -C 22 double bond in the steroidal triglycoside aglycone containing a C-12 hydroxyl contribute to the selective cytotoxicity to the SF-268 and NCI-H460 cell lines, respectively.

Plant material
The bulbs of Allium Macrostemon Bunge were purchased from Liaoning Province, P. R. China, and identified by Professor Qishi Sun (Department of Pharmacognosy, Shenyang Pharmaceutical University). The voucher specimen (No.203554) has been deposited at the Department of Natural Product Chemistry, Shenyang Pharmaceutical University, P. R. China.

Extraction and isolation
The dried bulbs of Allium Macrotemon Bunge (6 kg) were extracted twice with 60% ethanol for 2 hours each. The alcoholic extract was concentrated under reduced pressure, suspended in water and then passed through Diaion HP-20 column using EtOH-H 2 O gradient system (0-100%). The 60% EtOH eluate fraction (100 g), which was subjected to silica gel column chromatography with   Table 2.  Table 3.

Acid hydrolysis of saponins
Each saponin (5 mg) was heated in an ampoule with aq. 15% HCl (5 mL) at 110 °C for 2 h. The aglycon was extracted with dichloromethane three times and the aqueous residue was evaporated under reduced pressure. Then, pyridine (1 mL) and NH 2 OH·HCl (2 mg) were added to the residue, and the mixture was heated at 100 °C for 1 h. After cooling, Ac 2 O (0.5 mL) was added and the mixtures were heated at 100 °C for 1 h. The reaction mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC-MS using standard aldononitrile peracetates as reference samples.

Cytotoxicity assay
1.0×10 4 MCF-7, NCI-H460, SF-268 and HepG2 cells were seeded in 96 well tissue culture plates and treated with the two compounds on different concentration for 48 h. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent (5 mg/mL in PBS, 10 μL) was added to each well and incubated for 4 h. After that, the suspended liquid was poured out and DMSO (100 μL) added to each well and swirled gently. Finally, the plate cover was removed and the absorbance in each well measured at 570 nm in a micro titer plate reader [8].