A New Triterpenoid Saponin from Pulsatilla cernua

A new triterpenoid saponin was isolated from Pulsatilla cernua, along with eight known triterpenoids and triterpenoid glycosides. The new compound was identified as 3-O-β-d-glucopyranosyl-(1→4)-α-l-arabinopyranosyl-bayogenin-28-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl ester (1) on the basis of 1D, 2D-NMR techniques, including COSY, HMBC, and HMQC correlations, MS analysis, as well as chemical methods.


Introduction
Pulsatilla cernua (Thunb.) Bercht. et Opiz is widely distributed in Northeast China. The roots are used as a Traditional Chinese Medicine (TCM) for the treatment of amoebic and bacterial dysentery. Phytochemical studies on this plant were reported previously [1][2][3]. In the search for new and bioactive components from TCM, we investigated the roots of P. cernua. In the present paper, we report the isolation and structure elucidation of a new triterpenoid saponin from this source.

-O-α-L-arabinopyranosylhederagenin (4) [6], 3-O-β-D-glucopyranosyl-(1→4)-α-
g l u c o p y r a n o s y l -h e d e r a -g e n i n ( 8 ) [ 1 0 ] a n d 3 -O -β -D -g l u c o p y r a n o s y l ( 1 [11], The compounds 2, 4, 5, 7-9 were isolated for the first time from P. cernua. Herein, we describe the isolation and structure elucidation of the new compound.  H-12), and a signal at δ 3.15 (1H, dd, J = 3.0, 12.5 Hz, H-18) observed in the 1 H-NMR spectrum coupled with the information from the 13 C-NMR spectrum (six methyl group carbons at δ 15.1, 17.5, 17.8, 24.1, 26.7, and 33.2, and two olefinic carbons at δ 123.4 and 144.2) indicated that the aglycone possessed an olean-12-ene skeleton. Comparison of the 13 C-NMR data of this aglycone ( Table 1) with those of bayogenin (2α, 3β, 23-trihydroxyolean-12-en-28-oic acid) [5], showed that the signal for C-3 of 1 was shifted significantly downfield by +4.4 ppm to 83.0, and the C-28 signal was shifted upfield by -2.0 ppm to 176.6, while the other signals were almost identical, indicating that the aglycon of 1 was indeed bayogenin.  [10]. In a comparison of the 13 C-NMR signals for sugar moieties of 1 with those of the known saponin of leontoside ( [10], all signals due to the sugar moieties of 1 were almost superimposable with those of leontoside, indicating the sugar moieties of 1 was same as those of the latter, so the 3-hydroxy and 28-carbonyl groups carried the same disaccharide chain and trisaccharide chain, respectively. Consequently, compound 1 should be a bisdesmosidic saponin in which the disaccharide chain of arabinose and glucose was bound to the aglycone by a glycosidic linkage at C-3, while a trisaccharide chain of glucose, glucose, and rhamnose was bound by a glycosidic ester linkage at C-28. The 1 H-and 13 C-NMR spectrum of 1 exhibited five sugar anomeric protons at  Table 1). The methyl carbon signal at δ 18.7 and the doublet methyl proton signal at δ1.71 (3H, d, J = 6.0 Hz, rha H-6) indicating the presence of one 6-deoxysugar. These coupling constants indicated that the glycosidic linkage of arabinose, rhamnose were α configuration, and those of glucose were β configuration [12,13]. The 1 H-and 13 C-NMR signals for the aglycone and sugar moieties of 1 was assigned based on the 1D and 2D-NMR spectra

General
The melting point was determined on a Kofler-microscope apparatus and is uncorrected. The IR spectra were measured on a Y-Zoom scroll Fourier Transform infrared (FTIR) spectrometer using KBr discs. The ESI-MS was recorded on LCQ-1700 ESI-MS instrument made by the Finnigan (USA). The NMR spectra were obtained on Bruker AM-500 instrument, using TMS as internal standard. HPLC (600E HPLC, Waters, USA) was performed using a ODS column (Shim-park PREF-ODS, 250 × 4.6 mm). Column chromatography was performed on silica gel (200-300 mesh, Qingdao Oceanic Chemical Industry China) and ODS reversed silica gel (250 × 25 mm, Nacalai Tesque, Kyoto, Japan). Macroporous resin D 101 made in Nankai University, Tianjin. TLC was conducted on silica gel 60 F 254 (Merck). Spots were detected after spraying with 10% H 2 SO 4 .

Plant Material
The

Acid Hydrolysis of 1
Compound 1 (10 mg) was heated with 2 M HCl-MeOH (10 mL) under reflux for 3h. The reaction mixture was diluted with H 2 O and extracted with CHCl 3 . The water layer was neutralized with Na 2 CO 3 , concentrated, and subjected to TLC analysis with authentic glucose, arabinose, rhamnose, and developed with CH 2 Cl 2 -MeOH-H 2 O (15 ‫׃‬ ‫.)1׃6‬ Detection was carried out with aniline phthalate spray.