Saponins with neuroprotective effects from the roots of Pulsatilla cernua

Article abstract of DOI:10.3390/molecules17055520

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

Full text of DOI:10.3390/molecules17055520

Molecules 2012, 17, 5520-5531; doi:10.3390/molecules17055520
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Saponins with Neuroprotective Effects from the Roots of
Pulsatilla cernua
Jian-Yu Liu 1,2, Ying-Li Guan 3, Li-Bo Zou 4, Yi-Xia Gong 1,2, Hui-Ming Hua 1,5, Yong-Nan Xu 1,2,*,
Hui Zhang 1,2, Zong-Gui Yu 1,2 and Wen-Hao Fan 1,2
1
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; E-Mail: burningice@126.com (J.-Y.L.)
2
School of Pharmaceutical Engineer, Shenyang Pharmaceutical University, Shenyang 110016, China
3
Department of Pharmarcy and Food Science, Tonghua Normal University, Tonghua 134000, China
4
Department of Pharmacology, School of Life Science and Biopharmaceutics,
Shenyang Pharmaceutical University, Shenyang 110016, China
5
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China
* Author to whom correspondence should be addressed; E-Mail: ynxucn@yahoo.com.cn;
Tel.: +86-24-23986445.
Received: 1 April 2012; in revised form: 3 May 2012 / Accepted: 4 May 2012 /
Published: 9 May 2012
Abstract: 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.
Keywords: Pulsatilla cernua; saponin; neuroprotective effect; Alzheimer’s disease;
β-amyloid
1. Introduction
Pulsatilla cernua (Thunb.) Bercht. et Opiz. (Ranunculaceae) is a traditional medicinal plant in
northeastern China and Korea. The roots are used for the treatment of amoebic dysentery, malaria and

Molecules 2012, 17
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chills [1]. Previous phytochemical investigations on this plant have reported a number of triterpenoid
saponins [1–9]. Modern researches have shown that the chemical components of P. cernua had strong
biological activities, and especially the saponin-enriched fraction with some active components
showing remarkable effects in the treatment of Alzheimer’s disease (AD) [10,11]. In this paper, the
n-BuOH-soluble fraction of this plant was investigated to yield four new saponins, together with
six known saponins which were identified as cussonoside B (5) [12], pulsatiloside C (6) [13],
hederacochiside C (7) [1,7], patrinia saponin H3 (8) [7], aralia saponin 3 (9) [14], bayogenin 28-O-α-
L- rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester (10) [15] (Figure 1).
This paper describes the structural determination of the four new saponins and the neuroprotective
effect of some of these compounds against the cytotoxicity induced by β-amyloid25–35 (Aβ25–35) on
human neuroblastoma SH-SY5Y cells.
Figure 1. Chemical structures of compounds 1–10.
2. 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
3
configurations for L-arabinose were determined by their JH1,H2 coupling constants of 7–8 Hz. The
α-anomeric configuration of L-rhamnose was judged by the chemical shift of C-5 (δC 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 molecular formula of 1 was determined as C77H126O40 from HR-TOF-MS (m/z 1689.7773
13
[M−H]−, calc. 1689.7752). The compound displayed 77 carbon signals in the C-NMR spectrum, of
which 30 were assigned to the aglycone and the remaining 47 to the sugar moieties. The seven methyl
carbon signals at δC 15.4, 17.0, 17.2, 23.5, 25.8, 28.0 and 32.9, and the two olefinic carbon signals at
1
δC 122.9 and 144.2, coupled with the H-NMR data, seven tertiary methyl proton singlets at δH 0.82,

Molecules 2012, 17
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0.84, 0.84, 1.03, 1.12, 1.19, and 1.26, and a broad triplet-like olefinic proton signal at δH 5.35 (brs),
indicated that the aglycone possessed an olean-12-ene skeleton. C18 configuration was recognized as
1
H18β by the chemical shift of C12, C13 and C16 due to the γ-gauche interactions [16]. The H-NMR
spectrum showed eight anomeric proton signals at δH 4.61 (1H, d, J = 6.0 Hz), 4.96 (1H, d, J = 7.2 Hz),
5.10 (1H, d, J = 7.8 Hz), 5.15 (1H, d, J = 7.8 Hz) 5.42 (1H, d, J = 7.8 Hz), 5.84 (1H, brs), 6.19 (1H,
brs), 6.22 (1H, d, J = 7.8 Hz), as well as two methyl doublets of rhamnose at δH 1.52 (3H, d,
J = 6.0 Hz) and 1.66 (3H, d, J = 6.0 Hz), and the corresponding anomeric carbon signals at δC 95.4,
101.3, 102.5, 104.6 104.8, 105.1, 106.3, 106.4, respectively (Table 1). The chemical shifts of δC 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 δ 4.61 (1H, d, J = 6.0 Hz, ara
H-1) and δ 88.4 (C-3), δ 5.10 (1H, d, J = 7.8 Hz, glc H-1) and δ 79.9 (ara-4), δ 6.19 (1H, brs, rha H-1)
and δ 75.7 (ara-2), δ5.42 (1H, d, J = 7.8 Hz, glc′ H-1) and δ 83.0 (rha-3), δ 5.15 (1H, d, J = 7.8 Hz,
glc″″ H-1) and δ 80.9 (glc′-4), and the linkage at C-28 was established from the HMBC correlations
between δ 6.22 (1H, d, J = 7.8 Hz, glc″ H-1) and δ 176.5 (C-28), 4.96 (1H, d, J = 7.2 Hz, glc″′ H-1)
and δ 68.9 (glc″-6), 5.84 (1H, brs, rha′ H-1) and δ 77.9 (glc″′-4) (Figure 2). Based on the above
evidence, the structure of 1 was determined to be oleanolic acid 3-O-β-D-glucopyranosyl(1→4)-β-D-
gluco-pyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-
28-O- α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
The molecular formula of 2 was determined as C77H126O39 from HR-TOF-MS (m/z 1673.7813
[M−H]−, calc. 1673.7803). The NMR spectra of aglycone were in good agreement with those of 1. The
1H-NMR spectrum showed eight anomeric proton signals at δH 4.76 (1H，d, J = 6.0 Hz), 4.96 (1H,
overlapped), 5.11 (1H, d, J = 7.2 Hz), 5.39 (1H, brs), 5.40 (1H, d, J = 7.8 Hz), 5.85 (1H, brs), 6.21
(1H，overlapped), 6.23 (1H, brs), as well as three methyl doublets of rhamnose at δH 1.50 (3H, d,
J = 6.0 Hz), 1.55 (3H, d, J = 6.0 Hz), 1.67 (3H, d, J = 6.0 Hz), and the corresponding carbon signals at
δC 95.4, 101.4, 102.6, 102.7, 104.8, 104.9, 105.2, 106.4, respectively (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-
glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[α-L-rhamnopyranosyl(1→6)-β-D-glucopyranosyl
(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-
glucopyranosyl ester.
The molecular formula of 3 was determined as C77H126O40 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 (δH 1.27 and δC 28.2) in 2 to hydroxymethyl group (δH 3.89, 4.21 and δC 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.

Molecules 2012, 17
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Table 1. Spectroscopic data of 1 and 4 (sugar moieties, δ in ppm, J in Hz) in pyridine-d5.
1
4
1
4
δC
δH
δC
δH
δC
δH
δC
δH
C3-
glc″″-1
104.8
74.5
78.0
71.2
78.2
5.15(1H,d,7.8)
3.95(1H,m)
4.20(1H,m)
4.89(1H,brs)
4.10(1H,m)
4.27(1H,m),
4.53(1H,m)
ara-1
105.1
75.7
73.8
79.1
4.61(1H,d,6.0)
4.15(1H,m)
104.7
75.5
74.1
80.7
4.91(1H,d,6.0)
4.42(1H,m)
4.55(1H,m)
4.30(1H,m)
2
3
4
5
2
3
4
4.33(1H,m)
4.74(1H,dd,2.4,9.6)
5
65.1
3.69(1H,d,11), 4.40(1H,m)
66.4
3.62(1H,d,6.0), 4.20(1H,m)
6
62.2
glc-1
106.4
75.3
78.2
71.0
78.5
62.3
5.10(1H,d,7.8)
4.15(1H,m)
105.0
74.6
78.6
71.6
78.4
62.5
4.99(1H,d,7.8)
4.02(1H,m)
C28-
2
3
4
5
6
glc″-1
95.4
74.3
78.5
70.6
77.8
6.22(1H,d,7.8)
4.15(1H,m)
4.40(1H,m)
4.20(1H,m)
3.90(1H,m)
4.34(1H,m),
4.63(1H,m)
4.96(1H,d,7.2)
3.95(1H,m)
4.15(1H,m)
4.10(1H,m)
3.61(1H,d,9.0)
4.05(1H,m),
4.17(1H,m)
5.84(1H,brs)
4.69(1H,m)
4.50(1H,m)
4.30(1H,m)
4.31(1H,m)
1.66(3H,d,6.0)
95.8
74.2
78.9
71.0
78.4
6.14(1H,d,7.8)
4.15(1H,m)
4.40(1H,m)
4.33(1H,m)
3.92(1H,m)
4.33(1H,m),
4.82(1H,m)
4.90(1H,d,7.8)
3.93(1H,m)
4.13(1H,m)
4.09(1H,m)
3.57(1H,d,9.0)
4.05(1H,m),
4.15(1H,m)
5.76(1H,brs)
4.65(1H,m)
4.55(1H,m)
4.35(1H,m)
4.93(1H,m)
1.61(3H,d,6.0)
4.19(1H,m)
4.20(1H,m)
2
3
4
5
4.85(1H,m)
4.20(1H,m)
3.90(1H,m)
3.90(1H,m)
4.37(1H,m), 4.53(1H,m)
4.25(1H,m), 4.45(1H,m)
rha-1
101.3
6.19(1H,brs)
101.3
6.13(1H,brs)
6
69.0
69.4
2
3
4
5
6
71.4
83.0
72.7
69.5
18.3
4.90(1H,m)
4.74(1H,dd,2.4,9.6)
4.50(1H,m)
72.0
83.8
73.2
69.6
18.6
4.65(1H,m)
4.69(1H,dd,6.0,12.0)
4.35(1H,m)
glc″′-1
104.6
75.1
76.5
78.0
76.9
103.1
74.9
76.7
78.2
77.3
2
3
4
5
4.95(1H,m)
4.65(1H,m)
1.52(3H,d,6.0)
1.50(3H,d,6.0)
glc′-1
106.3
5.42(1H,d,7.8)
106.7
5.11(1H,d,7.8)
6
61.0
61.5
2
3
4
5
6
75.2
76.5
81.0
76.3
61.7
3.95(1H,m)
4.30(1H,m)
75.8
76.7
69.7
77.0
62.0
3.90(1H,m)
4.30(1H,m)
rha′-1
102.5
72.3
72.5
73.7
70.1
18.3
102.4
72.7
72.9
74.1
70.5
18.7
2
3
4
5
6
4.35(1H,m)
4.40(1H,m)
4.10(1H,m)
4.10(1H,m)
3.84(1H,m), 4.43(1H,m)
3.84(1H,m), 4.43(1H,m)

Molecules 2012, 17
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Table 2. Spectroscopic data of 2 and 3 (sugar moieties, δ in ppm, J in Hz) in pyridine-d5.
2
3
2
3
δC
δH
δC
δH
δC
δH
δC
δH
C3-
glc′-1
106.4
75.4
78.2
71.7
78.2
5.40(1H,d,7.8)
3.90(1H,m)
4.19(1H,m)
4.85(1H,m)
4.15(1H,m)
4.38(1H,m),
4.49(1H,m)
106.7
75.4
78.2
71.8
78.2
5.39(1H,O)
3.90(1H,m)
4.20(1H,m)
4.25(1H,m)
4.15(1H,m)
ara-1
105.2
75.2
74.0
81.9
4.76(1H,d,6.0)
4.49(1H,m)
4.29(1H,m)
4.22(1H,m)
3.75(1H,d,30),
4.16(1H,m)
5.11(1H,d,7.2)
4.10(1H,m)
4.19(1H,m)
4.40(1H,m)
4.00(1H,m)
3.91(1H,m),
4.58(1H,m)
104.9
75.3
74.0
82.0
4.95(1H,d,6.0)
4.49(1H,m)
4.25(1H,m)
4.19(1H,m)
2
3
4
5
2
3
4
5
65.8
66.6
3.60(1H,m), 4.18(1H,m)
6
61.8
61.8
4.36(1H,m), 4.42(1H,m)
glc-1
104.9
73.9
78.2
71.2
77.0
105.1
72.6
78.2
71.0
76.8
5.06(1H,d,7.8)
4.49(1H,m)
4.15(1H,m)
4.25(1H,m)
4.00(1H,m)
C28-
2
3
4
5
glc″-1
95.6
74.7
78.6
70.8
6.21(1H,O)
4.15(1H,m)
4.39(1H,m)
4.25(1H,m)
95.7
74.9
78.8
70.8
6.20(1H,d,8.4)
4.15(1H,m)
4.45(1H,m)
4.25(1H,m)
2
3
4
6
68.5
68.6
3.90(1H,m), 4.57(1H,d,9.0)
5.37(1H,brs)
5
6
78.0
69.1
4.00(1H,m)
78.2
69.2
3.90(1H,m)
4.32(1H,m),
4.60(1H,m)
4.96(1H,O)
3.95(1H,m)
4.13(1H,m)
4.10(1H,m)
3.61(1H,d,12)
4.05(1H,m),
4.17(1H,m)
5.85(1H,brs)
4.63(1H,m)
4.49(1H,m)
4.30(1H,m)
4.90(1H,m)
1.67(3H,d,6.0)
rha″-1
102.7
5.39(1H,brs)
102.8
4.29(1H,m), 4.63(1H,m)
2
3
4
5
6
71.8
71.8
73.9
69.8
18.4
3.90(1H,m)
4.85(1H,m)
4.29(1H,m)
4.25(1H,m)
1.49(3H,d,6.0)
72.0
72.0
73.1
69.9
18.5
4.85(1H,m)
4.65(1H,m)
4.44(1H,m)
4.21(1H,m)
1.51(3H,d,6.0)
glc″′-1
104.8
75.2
76.4
78.1
77.1
104.9
75.3
76.5
78.2
77.2
4.92(1H,O)
3.95(1H,m)
4.15(1H,m)
4.00(1H,m)
3.62(1H,m)
2
3
4
5
rha-1
101.4
6.23(1H,brs)
101.4
6.23(1H,brs)
6
61.2
61.3
4.05(1H,m), 4.17(1H,m)
2
3
4
5
6
71.6
83.5
73.0
69.6
18.5
4.25(1H,m)
4.70(1H,m)
4.43(1H,m)
4.59(1H,m)
1.54(3H,d,6.0)
71.7
83.5
72.8
69.7
18.7
3.90(1H,m)
4.75(1H,dd,3.0,9.6)
4.17(1H,m)
rha′-1
102.7
72.5
72.7
73.8
70.2
18.4
102.9
72.6
72.8
73.9
70.4
18.6
5.81(1H,brs)
4.65(1H,m)
4.50(1H,m)
4.28(1H,m)
4.91(1H,m)
1.66(3H,d,6.0)
2
3
4
5
6
4.06(1H,m)
1.55(3H,d,6.0)

Molecules 2012, 17
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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 3-O-β-D-
glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[α-L-rhamnopyranosyl(1→2)-β-D-glucopyranosyl
(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-
glucopyranosyl ester.
The molecular formula of 4 was determined as C71H114O36 from HR-TOF-MS (m/z 1541.6973
[M−H]−, calc. 1541.7017). The NMR spectra of aglycone were also similar to those of 2, apart from
the change of the methyl group (δH 1.27 and δC 28.2) in 2 to aldehyde group (δH 9.67 and δC 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). Thus, the structure of 4 was determined to be gypsogenin 3-O-β-
D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-
28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Figure 2. Key HMBC correlations (from H to C) of compound 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 Aβ25–35, a peptide comprising 11 residues within Aβ1–42
,
aggregates and retains the neurotoxin activities just like the full-length Aβ. Therefore Aβ25–35 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 Aβ25–35 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

Molecules 2012, 17
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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 Aβ25–35 on SH-SY5Y cell and can be further
investigated.
Figure 3. Effect of compounds 1, 4, 7 and 8 on cell ciability in Aβ25–35-induced cytotoxicity.
Compounds 1, 4, 7 and 8 reduced Aβ25–35-induced cytotoxicity in human SH-SY5Y cells. The
cultured cells were pre-incubated with different compounds for 18 h, and exposed to 20 μM Aβ25–35 for
an additional 30 h, then 15 μL of MTT stock solution was added to the culture medium for 4 h at 37 °C.
Cell viability was determined by measuring MTT reduction. Results are shown as the mean ± SEM
and represent six independent experiments. *p < 0.05, **p < 0.01 vs. model group (treated with Aβ25–35);
# p < 0.05 vs. control group.
3. Experimental
3.1. General
The NMR spectra were measured in pyridine-d5, 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 GF254
plates (Merck, Germany) and detected by spraying with 10% H2SO4 followed heating. GC analyses
were performed using an Agilent GC 6890 instrument on an HP-5 column (320 μm × 30 m, 0.25 μm).

Molecules 2012, 17
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3.2. 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).
3.3. 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, CH2Cl2, 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 CHCl3-MeOH-H2O (100:10:1→100:60:10) to give
three fractions (Fr.1 to Fr.3), and the 70% eluting fraction was likewise treated with a gradient of
CHCl3-MeOH-H2O (100:10:1→100:30:4) to give one fraction (Fr.4). Fr.3 was then subjected to HPLC
with MeOH-CH3CN-H2O (3:2:3) as eluent and each subfraction was further purified on recycling
preparative HPLC with MeOH-CH3CN (3:2) to yield 1 (60 mg), 3 (5.6 mg), 4 (8.6 mg), 5 (15 mg), 6
(36 mg), 7 (27 mg), 8 (18 mg), 9 (10 mg) and 10 (5 mg). Fr.4 was then subjected to HPLC with
MeOH-CH3CN-H2O (3:2:1) and further purified on recycling preparative HPLC with MeOH-CH3CN
(3:2) to yield 2 (6 mg).
3.4. Spectral Data
Oleanolic acid 3-O-β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl
(1→2)[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-
glucopyranosyl(1→6)-β-D-glucopyranosyl ester (1). White amorphous powder; mp. 270–273 °C;
1
HR-TOF-MS m/z 1689.7773 [M−H]− (calc. for C77H126O40, m/z 1689.7752); H-NMR of aglycone δ:
0.89 (1H, m, H-1a), 1.45 (1H, m, H-1b), 1.85 (1H, m, H-2a), 2.05 (1H, m, H-2b), 3.19 (1H, dd, J = 3.6,
11.0 Hz, H-3), 0.72 (1H, d, J = 12.0 Hz, H-5), 1.60 (2H, m, H-6), 1.29 (1H, m, H-7a), 1.40 (1H, m,
H-7b), 1.61 (1H, m, H-9), 0.88 (2H, m, H-11), 5.35 (1H, s, H-12), 1.16 (1H, m, H-15a), 2.27 (1H, m,
H-15b), 1.90 (1H, m, H-16a), 2.05 (1H, m, H-16b), 3.13 (1H, d, J = 12.0 Hz, H-18), 1.21 (1H, m,
H-19a), 1.73 (1H, m, H-19b), 0.90 (1H, m, H-21a), 1.09 (1H, m, H-21b), 1.75 (1H, m, H-22a), 1.85
(1H, m, H-22b), 1.26 (3H, s, H-23), 1.12 (3H, s, H-24), 0.84 (3H, s, H-25), 1.03 (3H, s, H-26), 1.20
(3H, s, H-27), 0.84 (3H, s, H-29), 0.82 (3H, s, H-30); 13C-NMR of aglycone δ: 38.5 (C-1), 26.5 (C-2),
88.3 (C-3), 39.3 (C-4), 55.8 (C-5), 18.3 (C-6), 32.9 (C-7), 39.7 (C-8), 47.8 (C-9), 36.8 (C-10), 23.6
(C-11), 122.6 (C-12), 144.9 (C-13), 41.9 (C-14), 27.9 (C-15), 23.1 (C-16), 46.8 (C-17), 41.4 (C-18),
46.0 (C-19), 30.5 (C-20), 33.8 (C-21), 32.3 (C-22), 28.0 (C-23), 16.9 (C-24), 15.4 (C-25), 17.2 (C-26),
1
13
25.9 (C-27), 176.5 (C-28), 32.9 (C-29), 23.5 (C-30); H-NMR and C-NMR of sugar moieties, see
Table 1.

Molecules 2012, 17
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Oleanolic acid 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[α-L-rhamnopyranosyl
(1→6)-β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-
glucopyranosyl(1→6)-β-D-glucopyranosyl ester (2). White amorphous powder; HR-TOF-MS m/z
1673.7813 [M−H]− (calc. for C77H126O39, m/z 1673.7803); 1H-NMR of aglycone δ: 0.89 (1H, m, H-1a),
1.45 (1H, m, H-1b), 1.83 (1H, m, H-2a), 2.05 (1H, m, H-2b), 3.24 (1H, d, J = 12.0 Hz, H-3), 0.74 (1H,
m, H-5), 1.60 (2H, m, H-6), 1.25 (1H, m, H-7a), 1.40 (1H, m, H-7b), 1.58 (1H, m, H-9), 0.88 (2H, m,
H-11), 5.34 (1H, s, H-12), 1.09 (1H, m, H-15a), 2.27 (1H, m, H-15b), 1.91 (1H, m, H-16a), 2.02 (1H,
m, H-16b), 3.12 (1H, d, J = 18.0 Hz, H-18), 1.19 (1H, m, H-19a), 1.69 (1H, m, H-19b), 1.06 (1H, m,
H-21a), 1.29 (1H, m, H-21b), 1.69 (1H, m, H-22a), 1.90 (1H, m, H-22b), 1.27 (3H, s, H-23), 1.13 (3H,
s, H-24), 0.84 (3H, s, H-25), 1.03 (3H, s, H-26), 1.20 (3H, s, H-27), 0.84 (3H, s, H-29), 0.83 (3H, s,
H-30); 13C-NMR of aglycone δ: 38.8 (C-1), 26.5 (C-2), 88.5 (C-3), 39.5 (C-4), 55.9 (C-5), 18.4 (C-6),
33.0 (C-7), 39.8 (C-8), 47.9 (C-9), 36.9 (C-10), 23.7 (C-11), 122.6 (C-12), 144.9 (C-13), 42.0 (C-14),
28.1 (C-15), 23.1 (C-16), 46.9 (C-17), 41.6 (C-18), 46.1 (C-19), 30.7 (C-20), 33.9 (C-21), 32.4 (C-22),
28.2 (C-23), 17.1 (C-24), 15.6 (C-25), 17.4 (C-26), 26.0 (C-27), 176.5 (C-28), 33.0 (C-29), 23.6 (C-30);
1H-NMR and 13C-NMR of sugar moieties, see Table 2.
Hederagenin 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)[α-L-rhamnopyranosyl(1→2)-
β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl
(1→6)-β-D-glucopyranosyl ester (3). White amorphous powder; HR-TOF-MS m/z 1689.7736 [M−H]−
(calc. for C77H126O40, m/z 1689.7752); 1H-NMR of aglycone δ: 1.02 (1H, m, H-1a), 1.50 (1H, m, H-1b),
1.95 (1H, m, H-2a), 2.19 (1H, m, H-2b), 4.21 (1H, m, H-3), 1.69 (1H, m, H-5), 1.60 (2H, m, H-6), 1.20
(1H, m, H-7a), 1.52 (1H, m, H-7b), 1.71 (1H, m, H-9), 1.90 (2H, m, H-11), 5.35 (1H, s, H-12), 1.15
(1H, m, H-15a), 1.65 (1H, m, H-15b), 1.95 (1H, m, H-16a), 1.99 (1H, m, H-16b), 3.12 (1H, dd, J = 3.6,
13.0 Hz, H-18), 1.16 (1H, m, H-19a), 1.66 (1H, m, H-19b), 1.05 (1H, m, H-21a), 1.22 (1H, m, H-21b),
1.70 (1H, m, H-22a), 1.80 (1H, m, H-22b), 3.89 (1H, m, H-23a), 4.23 (1H, m, H-23b), 1.10 (3H, s,
H-24), 0.94 (3H, s, H-25), 1.06 (3H, s, H-26), 1.14 (3H, s, H-27), 0.82 (3H, s, H-29), 0.84 (3H, s,
H-30); 13C-NMR of aglycone δ: 39.0 (C-1), 26.5 (C-2), 81.1 (C-3), 43.6 (C-4), 47.6 (C-5), 18.1 (C-6),
32.7 (C-7), 39.9 (C-8), 48.2 (C-9), 36.9 (C-10), 23.9 (C-11), 122.5 (C-12), 144.8 (C-13), 42.1 (C-14),
28.3 (C-15), 23.3 (C-16), 47.0 (C-17), 41.7 (C-18), 46.2 (C-19), 30.8 (C-20), 34.0 (C-21), 32.5 (C-22),
64.0 (C-23), 14.3 (C-24), 16.2 (C-25), 17.5 (C-26), 26.1 (C-27), 176.5 (C-28), 33.1 (C-29), 23.7 (C-30);
1H-NMR and 13C-NMR of sugar moieties, see Table 2.
Gypsogenin 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2) [β-D-glucopyranosyl(1→4)]-
α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl
ester (4). White amorphous powder; HR-TOF-MS m/z 1541.6973 [M−H]− (calc. for C71H114O36, m/z
1541.7017); 1H-NMR of aglycone δ: 1.27 (1H, m, H-1a), 1.93 (1H, m, H-1b), 1.95 (1H, m, H-2a), 2.17
(1H, m, H-2b), 4.70 (1H, m, H-3), 1.59 (1H, m, H-5), 1.12 (1H, m, H-6a), 1.75 (1H, m, H-6b), 1.49
(1H, m, H-7a), 1.75 (1H, m, H-7b), 1.93 (1H, m, H-9), 2.17 (2H, m, H-11), 5.30 (1H, s, H-12), 1.22
(1H, m, H-15a), 2.01 (1H, m, H-15b), 2.02 (1H, m, H-16a), 2.20 (1H, m, H-16b), 3.08 (1H, dd, J = 6.0,
12.0 Hz, H-18), 1.29 (1H, m, H-19a), 1.79 (1H, m, H-19b), 1.41 (1H, m, H-21a), 1.60 (1H, m, H-21b),
1.82 (1H, m, H-22a), 1.89 (1H, m, H-22b), 9.67 (1H, s, H-23), 1.39 (3H, s, H-24), 0.81 (3H, s, H-25),
0.95 (3H, s, H-26), 1.13 (3H, s, H-27), 0.79 (3H, s, H-29), 0.81 (3H, s, H-30); 13C-NMR of aglycone δ:

Molecules 2012, 17
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38.5 (C-1), 25.7 (C-2), 83.8 (C-3), 55.8 (C-4), 48.1 (C-5), 20.8 (C-6), 32.7 (C-7), 40.3 (C-8), 48.5
(C-9), 36.3 (C-10), 23.9 (C-11), 122.6 (C-12), 144.8 (C-13), 42.3 (C-14), 28.4 (C-15), 23.5 (C-16),
47.2 (C-17), 41.8 (C-18), 46.4 (C-19), 30.9 (C-20), 34.1 (C-21), 32.6 (C-22), 205.5 (C-23), 11.0 (C-24),
15.8 (C-25), 17.6 (C-26), 26.3 (C-27), 176.6 (C-28), 33.3 (C-29), 23.9 (C-30); 1H-NMR and 13C-NMR
of sugar moieties, see Table 1.
3.5. Acid Hydrolysis and GC Analysis
Compound 1 (4 mg) was treated with 1 M HCl (4 mL) at 90 °C for 2 h. The reaction mixture was
then extracted with CHCl3 (3 × 5 mL). Acid hydrolysis of 2–4 was performed likewise. Each
remaining aqueous layer was concentrated to dryness to give a residue and was dissolved in pyridine
(2 mL), and then L-cysteine methyl ester hydrochloride (2 mg) was added to the solution. Then the
mixture was heated at 60 °C for 1 h, and trimethylchlorosilane (0.5 mL) was added, followed by
heating at 60 °C for 30 min. Then, the solution was concentrated to dryness and taken up in water
(1 mL × 3), followed by extraction with n-hexane (1 mL × 3). The supernatant was subjected to GC
analysis under the following conditions: Agilent GC 6890 instrument equipped with FID (detection
temperature 280 °C). Column: HP-5 column (320 μm × 30 m, 0.25 μm). Column temperature:
160–200 °C with the rate of 4 °C/min, then kept for 5 min, and then 200–240 °C with the rate of
10 °C/min and kept for 10 min. The carrier gas was N2 (1.0 mL/min), split ratio 1/10, injection
temperature: 270 °C. Injection volume: 10 μL. The absolute configurations of the monosaccharides
were confirmed to be L-arabinose, L-rhamnose, and D-glucose by comparison of the retention times of
monosaccharide derivatives with those of standard samples: L-arabinose (12.67 min), L-rhamnose
(12.85 min), and D-glucose (14.41 min), respectively.
3.6. Cell Culture
Human neuroblastoma SH-SY5Y cells were cultured using DMEM/F12 culture (Gibco, USA),
supplemented with 10% (v/v) fetal bovine serum (Therom Scientific, USA), 100 U/mL penicillin and
100 μg/mL streptomycin at pH 7.4 and maintained in a humidified 5% CO2 atmosphere at 37 °C.
Experiment was carried out 24 h after cells were seeded.
3.7. Determination of Cell Viability
Cell viability was assessed using conventional 3-[4,5-dimethylthiazolo-2]-2,5-diphenyltetrazolium
bromide (MTT) assay. Aβ25–35 (Sigma-Aldrich, USA) was dissolved in distilled water and aged at
37 °C for 7 days before use. The cultured cells were pre-incubated with different compounds for 18 h
in 96-well plates, and exposed to 20 μM Aβ25–35 for an additional 30 h, then 15 μL of MTT stock
solution (5 mg/mL) (Sigma-Aldrich, USA) was added to the culture medium for 4 h at 37 °C. The
MTT formazan crystals were solubilized by 150 μL DMSO and the absorbance was measured at 492 nm
using a microplate reader (Tecan, Switzerland). Results were expressed as percentage of control.

Molecules 2012, 17
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3.8. Statistical Analysis
The results of cell viability were expressed as mean ± S.E.M. One-way ANOVA followed by
Dunnett’s t-test was performed to statistical analysis. p < 0.05 was considered statistically significant.
4. Conclusions
Four new oleanene-type triterpenoid saponins together with six known saponins were isolated from
the roots of P. cernua and their structures were elucidated on the basis of spectroscopic data, including
2D NMR spectra and chemical evidence. Among these compounds 9 was isolated for the first time
from this genus, and 5, 6, and 10 were reported from this plant for the first time. Furthermore, the
aglycone of 4 has not been reported from this genus before. Interestingly, other aglycones with
aldehyde groups at C-23 have not been obtained in this genus, either, which suggested that there might
be some specific enzyme system in P. cernua compared with other plants of this genus. Compounds 1,
4, 7 and 8 showed significant effect against the cytotoxicity induced by Aβ25–35 on SH-SY5Y cell
which suggested that these compounds might be good candidates for the prevention and treatment of
AD, and can be further investigated.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/5/5520/s1.
Acknowledgments
The authors are grateful to Ling-Yi Kong, Department of Natural Medicinal Chemistry,
China Pharmaceutical University for HR-TOF-MS experiments.
Conflict of Interest
The authors declare no conflict of interest.
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