Cytotoxic Triterpenoid Saponins from the Roots of Platycodon Grandiflorum

Bioguided fractionation of the ethanol extracts obtained from Platycodon grandiflorum roots led to isolation of two new triterpenoid saponins, characterized as


Structure Elucidation
The combined 25% EtOH and 75% EtOH fractions eluted from a D 101 macroporous resin column showed significant cytotoxic activity against human ECA-109 cells.Bioguided fractionation of the active fractions over silica gel eluting with a 95:5:0.5~50:50:5CHCl 3 -CH 3 OH-H 2 O gradient led to the isolation of two triterpenoid saponins 1-2 (Figure 1). 3) Compound 1 was obtained as a white powder with mp 222~223°C (CH 3 OH) and [α] 20 D : +11.76° (CH 3 OH; c 0.0170), that gave positive Libermann-Burchard and Molisch tests.The molecular formula was determined as C 36 H 58 O 13 , on the basis of its HRESIMS, which showed a [M+Na] + quasimolecular ion peak at m/z 721.3771 (calcd.for C 36 H 58 O 13 Na 721.3775).The spectral features and physicochemical properties suggested 1 to be a triterpenoid saponin.Five methyl groups (δ 1.09, 1.09, 1.34, 1.52 and 1.94) were observed in the 1 H-NMR spectrum.The 13 C-NMR spectrum of 1 (Table 1) showed 36 carbons, which were classified into five methyls, 11 methylenes, 12 methines and eight quaternary carbons by DEPT experiments.In addition, the 13 C-NMR spectrum displayed five sp 3 carbons at δ 18.4, 20.2, 21.0, 24.6 and 33.4,two oxygenated methylene carbons at δ 63.3 and 66.1, four oxygenated methine carbons at δ 69.2, 87.8, 66.1 and 72.1, and an oxygenated quaternary carbon at δ 92.8, attributable to the aglycone moiety.The NMR information thus indicated that 1 possessed a heptaoxygenated oleanane aglycone.D-glucose was detected by GC analysis after acidic hydrolysis and preparation of the corresponding thiazolidine derivative [33].The presence of a β-glucose moiety was concluded based on a typical doublet peak of the anomeric proton of the glucose at δ 5.14 (1H, d, J=7.7 Hz) in the 1 H-NMR, as well as the 13 C-NMR signals at δ 106.5, 75.4,78.9, 71.8, 78.9 and 62.7 of one glucose unit.In addition, the A-ring carbon signals of 1 were almost the same as those of a known compound, 3-O-β-D-glucopyranosylplatycodigenin methyl ester [10], indicating that the A-ring of 1 contained 2β,23,24-trihydroxyl and 3β-O-β-D-glucopyranosyl substituents.The 13 C-NMR signals at δ 92.8 and 178.1 revealed the existence of a lactone moiety in its structure.Based upon the above findings, it was deduced that 1 was a 3-O-β-D-glucopyranosyl-2β,3β,23,24-tetrahydroxy oleanane with another three oxygenated carbons, one (δ C 92.8) of which was due to the lactone moiety.The positions of the other substituents were determined by its 1 H-1 H COSY (Figure 2) and HMBC (Figure 3) spectra and literature data.The correlations between 25-CH 3 (δ H 1.52) and δ C 45.  32.0 (C-20), 24.6 (C-30) and 33.4 (C-29), respectively, indicated that these two methyl groups were located at the same carbon with the signal at δ C 32.0 (C-20).The proton signal at δ H 2.54 (H-21) had correlations with δ H 2.26 and 2.07 (both H-22) in the 1 H-1 H COSY spectrum.In addition, the correlations between δ H 2.07(H-22) and δ C 178.1(C-28) could also be observed in its HMBC spectrum, indicating that the carbonyl group of the lactone was located at C-28.Thus, it could be deduced that the lactone should be 28→13, a fact that was also be proved by comparison with the known oleanderolide-(lactone 28→13) [34].The proton signal at δ H 4.54, corresponding to an oxygenated methine carbon signal at δ C 66.1 in the HMQC experiment, was observed in the HMBC to correlate with both δ C 92.8(C-13) and 45.3(C-9), showing that this proton should be located at C-12 and a hydroxyl group substitutent must exist at C-12.Correlations between H-15 (δ H 2.43) and δ H 4.51 (H-16) corresponding to the oxygenated methine carbon signal at δ C 72.7 could be seen from the 1 H-1 H COSY data and were confirmed by the HMQC spectrum, indicating another hydroxyl group was located at C-16.
The configurations of the12,16-OH were determined by the NOESY spectrum (Figure 2).The presence of a NOESY effect between δ H 4.54 (H-12) and δ H 1.34 (26β-CH 3 ), but the absence of any NOESY effect between δ H 4.54 (H-12) and δ H 1.94 (27α-CH 3 ) indicated the α-configuration of 12-OH.In addition, the same result was also obtained in the NOESY spectrum for H-16 at δ H 4.51.Therefore, the structure of 1 was identified as 3-O-β-D-glucopyranosyl-2β,12α,16α,23,24-pentahydroxyoleanane-28(13)-lactone, which was a new compound.   C-NMR spectrum.In addition, all spectral features and physicochemical properties revealed that 2 might also be a triterpenoid saponin.The 13 C-NMR spectrum of 2 showed aglycone signals that were broadly similar to those of 1, except those attributable to the A-ring carbons (see Table 1).Consequently, 2 was assumed to have an aglycone with some differences in the A-ring compared to 1.Among the A-ring carbon signals of 2, it could be observed that they were almost identical to those of methyl 3-O-β-laminaribiosylpolygalacate [10], suggesting that 2 had the same A-ring moiety as the latter.Thus, the aglycone of 2 was deduced to be 2β,3β,12α,16α,23α-pentahydroxyoleanane-28( 13 1).These findings confirmed the presence of two β-glucose units in this molecule.D-glucose was also detected by GC analysis after acid hydrolysis and preparation of the thiazolidine derivatives.The spin-systems associated with saccharides were identified by a HMQC-TOCSY experiment with the aid of a 1 H-1 HCOSY spectrum.

Cytotoxic activity
The two new saponins identified in the present study were examined for their cytotoxic activity against the human Eca-109 cell line.Topotecan (IC 50 0.032µg/mL) was used as a standard in the cytotoxic assay.The saponins exhibited cytotoxicity against human Eca-109 and gave IC 50 values of 0.649µg/mL (1) and 0.503µg/mL (2), respectively.

General
All melting points were determined using a Fisher Johns apparatus and are uncorrected.Optical rotations were measured on a Perkin-Elmer 241 polarimeter.1D-and 2D-NMR spectra were recorded on a Bruker 400 spectrometer.The ESIMS and HRESIMS were recorded in a LCQ DECA XP plus spectrometer.An Agilent 1100 series HPLC was used with a Zorbax SB-C 18 preparative column.GC-MS was performed using a Shimadzu QP5050A instrument.Thin-layer chromatography employed precoated silica gel plates (Qingdao Haiyang).For column chromatography, silica gel (Qingdao Haiyang), D 101 macroporous resin (Tianjin Nankai) and Sephadex LH-20 (Pharmacia) were used.

Plant Material
Roots of P. grandiflorum were collected from Hangzhou in the Zhejiang province of China and identified by Dr Lin Zhang (Institute of Modern Traditional Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University).A voucher specimen was deposited at the Institute of Modern Traditional Chinese Medicine, College of Pharmaceutical sciences, Zhejiang University, P.R. China.

Acid Hydrolysis
Compound 1 and 2 (each 5 mg) were dissolved in water (100 mL) and 2M HCl (100 mL) and heated at 100 o C for 1 h.The water was passed through an Amberlite IRA-60E column (6×50 mm) and the eluates were concentrated.The residues were dissolved in pyridine (25 mL) and stirred with Dcysteine methyl ester (4.0 mg) for 1.5 h at 60 o C. To the reaction mixture, hexamethyldisilazane (10 mL) and trimethylsilyl chloride (10 mL) were added and the mixture was stirred for 30 min at 60 o C. The supernatants were then analyzed by GC [Column: DB-50, 25mm×30m, column temperature; 230 o C; carrier gas: N 2 , retention time D-Glc (16.4 min), L-Glc (16.0 min).From the new saponins, only D-glucose was detected.

Cytotoxicity Assays
Viability of ECA-109 cells in the presence or absence of experimental fractions or compounds was determined using the standard sulforhodamine B (SRB) assay as described previously [35].Briefly, assays were carried out in 96-well plates.4,000 cells/well were plated in media containing 5% FBS and were allowed to attach overnight.The cells were treated with 200 µL of media containing either 0.06% DMSO alone (control) or varying concentrations of test specimens dissolved in DMSO.The plates were incubated at 37 °C in a humidified incubator containing 5% CO 2 .The cells were fixed after 3 days by incubation in cold 50% TCA for 1 h at 4 °C in the dark.The media and TCA were removed and the plates were rinsed five times with water and then air-dried.The cells were stained by addition of 0.4% SRB (Sigma, St. Louis, MO) in 1% acetic acid (50 µL) for 5 min.The stain was removed and the cells were washed five times with 1% acetic acid, air-dried, and 150 µL of 10 mM unbuffered Tris was then added to each well to dissolve the dye.The plates were shaken for 5 min until the dye was uniformly distributed and then read on an Emax Precision Plate Reader (Molecular Devices, Sunnyvale, CA) at 570 nm.Media were used as the blank for these assays.

Figure 2 .
Figure 2. Key correlations in the 1 H-1 H COSY and NOESY spectra of 1.

Figure 3 .
Figure 3. Key correlations in the HMBC spectrum of 1.