Saponins with Neuroprotective Effects from the Roots of Pulsatilla cernua

Four new oleanene-type triterpenoid saponins together with six known saponins were isolated from the roots of Pulsatilla cernua and their structures were elucidated on the basis of spectroscopic data, including 2D NMR spectra and chemical evidence. Among these one of the aglycones (gypsogenin) is reported for the first time from this genus. Some of these compounds showed significant neuroprotective effects against the cytotoxicity induced by β-amyloid25–35 (Aβ25–35) on human neuroblastoma SH-SY5Y cells.


Results and Discussion
Compounds 1-4 were obtained as white amorphous powders. Acid hydrolysis of these compounds with 1 M HCl gave arabinose, rhamnose and glucose, which were identified by TLC comparison with authentic samples. The β-anomeric configurations for the D-glucose, and the α-anomeric configurations for L-arabinose were determined by their 3 J H1,H2 coupling constants of 7-8 Hz. The α-anomeric configuration of L-rhamnose was judged by the chemical shift of C-5 (δ C 69-70) [16]. The absolute configuration of the saccharides was determined to be D-for glucose, L-for rhamnose and arabinose by GC analysis of chiral derivatives in the hydrolysate of these compounds.
The  Table 1). The chemical shifts of δ C 88.3 (C-3) and 176.5 (C-28) revealed that 1 was a bidesmosidic saponin with a glycosidic linkage at C-3 through an O-heterosidic bond and at C-28 through an ester bond. The chemical shift of sugar moieties was comfirmed on the basis of the HSQC-TOCSY correlations. The linkage of the sugar moiety at C-3 of the aglycone was established from the HMBC correlations between  Table 2). The linkage of the sugar moieties at C-3 and C-28 of the aglycone was established from the HMBC correlations. Based on the above evidence, the structure of 2 was determined to be oleanolic acid 3-O-β-D- The molecular formula of 3 was determined as C 77 H 126 O 40 from HR-TOF-MS (m/z 1689.7736 [M−H] − , calc. 1689.7752). The NMR spectra were almost similar to those of 2, apart from the change of the methyl group (δ H 1.27 and δ C 28.2) in 2 to hydroxymethyl group (δ H 3.89, 4.21 and δ C 64.0) in 3, suggesting the hydrogen at C-23 in 2 was substituted by the hydroxyl group in 3. Thus, the aglycone of 3 was identified as hederagenin. Table 1. Spectroscopic data of 1 and 4 (sugar moieties, δ in ppm, J in Hz) in pyridine-d 5 .  Table 2. Spectroscopic data of 2 and 3 (sugar moieties, δ in ppm, J in Hz) in pyridine-d 5 . The NMR spectra of sugar moieties were in good agreement with those of 2 ( Table 2). The linkage of the sugar moieties at C-3 and C-28 of the aglycone was also established from the HMBC correlations, and the structure of 3 was determined to be hederagenin The NMR spectra of aglycone were also similar to those of 2, apart from the change of the methyl group (δ H 1.27 and δ C 28.2) in 2 to aldehyde group (δ H 9.67 and δ C 205.5) in 4. Thus, the aglycone of 4 was identified as gypsogenin. Compared with 2, the NMR spectra of sugar moieties were almost similar, only except for the absent of the terminal rhamnose on the sugar chain linked to C-3 of aglycone (Table 1)  One of the major pathological features of AD is the appearance of senile plaques characterized by extracellular aggregation of Aβ fibrils, 39 to 43 amino acid peptides derived from the amyloid precursor protein (APP). It has been shown that Aβ 25-35 , a peptide comprising 11 residues within Aβ 1-42 , aggregates and retains the neurotoxin activities just like the full-length Aβ. Therefore Aβ 25-35 was used in the experiment of this study [17,18]. Human neuroblastoma SH-SY5Y cell is a widely and extensively used target cell line in the assessment of neurotoxicity and neuroprotection.
Neuroprotective effects of compounds against the cytotoxicity induced by Aβ 25-35 on SH-SY5Y cell were tested by the MTT assay. Due to its knwon significant neuroprotective effects against Aβ [11], hederacochiside E was used as positive control. In the Aβ model group, the cell viability were decreased by ~20% compared with the control group. While in contrast, the cell viability of the groups treated by saponin 1, 4, 7 and 8 were increased by ~20% compared with the model group (p < 0.05, p < 0.01, p < 0.05, p < 0.05, respectively) at the highest concentration (100 μmol/L). In addition, the results of 1 and 4 were similar with the compound hederacochiside E (Figure 3). While other compounds showed no effects (data not shown). The results indicated that 1, 4, 7 and 8 showed significant effect against the cytotoxicity induced by Aβ 25-35 on SH-SY5Y cell and can be further investigated.

General
The NMR spectra were measured in pyridine-d 5 , on a Bruker AV600 instrument. ESI-MS spectra were recorded on Waters Quattro micro API LC/MS/MS spectrometer (Waters, USA). HR-TOF-MS spectra were performed on Agilent LC/MS spectrometer (Agilent, USA). HPLC was performed on JAI LC9103 Recycling preparative HPLC (Japan Analytical Industries, Japan) equipped with JAIGEL-ODS-AP-P column and JAIGEL-GS310 column using a JAI refractive index detector and a JAI UV-3702 detector with MultiChro 2000 workstation. TLC was performed on pre-coated GF 254 plates (Merck, Germany) and detected by spraying with 10% H 2 SO 4 followed heating. GC analyses were performed using an Agilent GC 6890 instrument on an HP-5 column (320 μm × 30 m, 0.25 μm).

Plant Material
The roots of P. cernua were collected in May 2007 at Qingyuan, Fushun, Liaoning, China, and authenticated by Professor Jin-Cai Lu (The School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen has been deposited in our laboratory (voucher No. pc-2007-001).

Extraction and Isolation
The air-dried and pulverized roots of P. cernua (5 kg) were extracted three times at 80 °C with 50% aqueous EtOH (20 L and 4 h each time), and then the extracts were combined and concentrated under reduced pressure at 60 °C in vacuo to give a residue (550 g), which was then suspended in water, and partitioned successively with petroleum ether, CH 2 Cl 2 , EtOAc and n-BuOH in the same volume (5 L) three times, respectively. The n-BuOH-soluble fraction (160.5 g) was subjected to column chromatography on D101 macroporous resin, and eluted with a gradient of aqueous EtOH (30%, 50%, 70%, 95% EtOH, respectively) to give four fractions. The 50% eluting fraction was subjected to silica gel chromatography, eluting with a gradient of  Table 1.  Table 2.  Table 2.