Platycoside N: A New Oleanane-Type Triterpenoid Saponin from the Roots of Platycodon grandiflorum

A new oleanane-type triterpenoid saponin, named platycoside N (1), together with six known saponins, was isolated from the roots of Platycodon grandiflorum. On the basis of acid hydrolysis, comprehensive spectroscopic data analyses and comparison with the spectral data of the known compounds, its structure was elucidated as 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-β-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. The six known compounds were platycodin D (2), deapioplatycodin D (3), platycodin D3 (4), deapio- platycodin D3 (5), platycoside E (6) and deapioplatycoside E (7).


General
ESI-MS (negative mode) measurements were carried out on an Agilent 1100 series LC/MSD Trap SL mass spectrometer. HR-ESI-MS (positive and negative modes) was analyzed on a Bruker FT-ICRMS spectrometer. IR spectra were recorded on an IR-47 spectrometer. NMR spectra were recorded on a Bruker Avance DRX 400 NMR spectrometer using TMS as internal standard, and chemical shifts δ were given in ppm. Silica gel (200-300 mesh) for column chromatography and silica gel G for TLC were purchased from Qingdao Marine Chemical Factory, Qingdao, China. AB-8 macroporous resin was purchased from Tianjin Nankai factory. Preparative HPLC was performed on a Waters 600 liquid chromatography instrument with a UV detector, monitored at 210 nm using a C18 column (Zorbax Eclipse XDB, 250 mm × 9 mm; 10 μm)

Plant material
The roots of P. grandiflorum were purchased at Changchun Guangfulu market in Changchun-city of Jilin province, China and identified by Prof. Yi-Nan Zheng, College of Chinese Material Medicine, Jilin Agricultural University. A voucher specimen (No.20050116) has been deposited in the herbarium of the same college.

Extraction and isolation
Dry and powdered roots of P. grandiflorum (2.0 Kg) were refluxed three times with 30 L of 70% methanol, 3 h each time. Extracts were concentrated, suspended in water and sequentially partitioned with ethyl acetate and n-butanol. The n-butanol fraction was subjected to macroporous resin AB-8 column and eluted sequentially with water, 30% ethanol and 70% ethanol. The 30% ethanol elution was repeatedly chromatographed on a reverse-phase column, and eluted with aqueous methanol, affording three fractions A-C.  Table 1.

Acid hydrolysis of 1
Compound 1 (2.0 mg) was refluxed with 4.0 M HCl (5.0 mL) for 1 h at 95 °C, and the reaction mixture was extracted with ethyl acetate. The aqueous layer was then adjusted to pH 7.0 with NaHCO 3 . After evaporating to dryness, the sugar mixture was dissolved in pyridine and developed on silica gel TLC [CHCl 3 -MeOH-H 2 O (7:3:0.5, lower phase), n-BuOH-AcOH-H 2 O (4:1:5, upper phase). Three spots were seen on the TLC after spraying with 4% α-naphthol-EtOH-5% H 2 SO 4 . Through comparison with authentic sugar standards (purchased from Sigma), it was found that compound 1 possessed D-glucose, L-rhamnose and L-arabinose units.

Conclusions
In summary, we have isolated a new oleanane-type triterpenoid saponin, named platycoside N (1), together with six known saponins from the roots of Platycodon grandiflorum.