Monoterpene glucosides from Paeonia lactiflora

Article abstract of DOI:10.1021/np8001783

Four new "cage-like" monoterpene glucosides (1-4) were isolated from Paeonia lactiflora. The structures of these compounds were established by spectroscopic methods, mainly 1D and 2D NMR, and mass spectrometric analysis. Compound 4 exhibited moderate cell

Full text of DOI:10.1021/np8001783

J. Nat. Prod. 2009, 72, 1321–1324
1321
Notes
Monoterpene Glucosides from Paeonia lactiflora
Hong-Bing Wang,† Wei-Feng Gu,† Wen-Jing Chu,† Sheng Zhang,‡ Xi-Can Tang,† and Guo-Wei Qin*,†
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 201203, People’s Republic of China, and Shanghai UniVersity of Traditional Chinese Medicine,
Shanghai 201203, People’s Republic of China
ReceiVed March 21, 2008
Four new “cage-like” monoterpene glucosides (1-4) were isolated from Paeonia lactiflora. The structures of these
compounds were established by spectroscopic methods, mainly 1D and 2D NMR, and mass spectrometric analysis.
Compound 4 exhibited moderate cell-protective activity against hydrogen peroxide-induced PC12 cell damage.
Paeonia lactiflora Pall. (Ranunculaceae) is a well-known orna-
mental plant with ornate flowers blooming in spring in mainland
China. Its dried whole roots and skin-free roots are officially listed
in the Chinese Pharmacopoeia (2005 edition) as the commonly used
traditional Chinese medicines “Chi-Shao” and “Bai-Shao”, respec-
tively, used for anti-inflammatory, analgesic, and sedative purposes.1
The chemical constituents of P. lactiflora have been investigated
in detail, resulting in the isolation of various monoterpene
compounds, among which paeoniflorin, the main constituent of the
roots, possesses a “cage-like” skeleton.2-4 Paeoniflorin and other
“cage-like” monoterpene derivatives show antihyperglycemic, anti-
inflammatory, antioxidant, and other biological activities.1,5,6 Pae-
oniflorin has been patented for the treatment of dementia and is
considered as a candidate compound for cognitive enhancement.7
In the course of a search for nootropic compounds from Chinese
medicinal plants, we have investigated the dried roots of P.
lactiflora. From an ethanol extract of the plant roots, four new
monoterpene glucosides were isolated and named paeonins A (1),
B (2), and C (3) and 8-debenzoylpaeonidanin (4). In this paper,
we describe the isolation and structural elucidation of compounds
1-4 as well as their in vitro cell-protective activities.
A 95% EtOH extract of the plant roots (5.0 kg) was extracted
with petroleum ether, ethyl acetate, and n-butanol, successively.
From a series of column chromatographic separations, 1 (12 mg),
2 (8 mg), 3 (13 mg), and 4 (5 mg) were obtained from the ethyl
acetate extract.
Paeonin A (1), a colorless oil, showed the molecular formula
C17H24O9, as determined by HRESIMS m/z 395.1286 [M + Na]+
1
(calcd for C17H24O9Na, 395.1318), together with the H and 13C
NMR data. The UV spectrum revealed an R,ꢀ-unsaturated ketone
at 252 nm. The IR spectrum displayed absorption bands for a
hydroxyl group (3365 cm-1), double bonds (1612, 1640 cm-1), and
a conjugated carbonyl group (1679 cm-1). Compound 1 was positive
in the Molish test. Hydrolysis of 1 with ꢀ-glucosidase yielded
1
glucose as a sugar moiety, as determined by co-HPTLC. The H
three methines [δ 3.29 (1H, dd, J ) 3.4, 2.6 Hz, H-4), δ 6.05 (1H,
q, J ) 1.3 Hz, H-2), δ 6.70 (1H, s)], and one glucose unit [δ 5.43
(1H, d, J ) 7.9 Hz, H-1′), 3.68 (1H, m, H-2′), 3.57 (1H, m, H-3′),
3.43 (1H, m, H-4′), 3.46 (1H, m, H-5′), 3.73 and 3.94 (2H, m,
H-6′)]. The 13C NMR data (Table 2) contained signals for two
methyls (one olefinic, one oxygenated), two methylenes (one
oxygenated), three methines (two olefinic), and four quaternary
carbons (one carbonyl, two olefinic, one semiketal), together with
a ꢀ-glucopyranosyl unit. These spectroscopic features suggested
that 1 is a monoterpene glucoside, and its monoterpene moiety
NMR data (Table 1) contained the signals for an olefinic methyl
[δ 2.06 (3H, d, J ) 1.3 Hz, H-10)], a methoxy [δ 3.14 (3H, s,
H-11)], two nonequivalent methylenes [δ 2.48 (1H, dd, J ) 13.2,
3.4 Hz, H-5R), 2.59 (1H, dd, J ) 13.2, 2.6 Hz, H-5ꢀ) and δ 3.69
(1H, d, J ) 11.9 Hz, H-8R), 3.94 (1H, d, J ) 11.9 Hz, H-8ꢀ)],
* To whom correspondence should be addressed. Tel: +86-21-50805853.
Fax: +86-21-50807088. E-mail: gwqin@mail.shcnc.ac.cn.
† Shanghai Institute of Materia Medica.
‡ Shanghai University of Traditional Chinese Medicine.
10.1021/np8001783 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/29/2009

1322 Journal of Natural Products, 2009, Vol. 72, No. 7
Notes
Figure 1. Key HMBC (HfC) correlations of compounds 1-4.
Paeonin C (3), an amorphous powder, showed the molecular
formula C17H24O9, on the basis of its HRESIMS (m/z 395.1326,
[M + Na]+). Hydrolysis of 3 with ꢀ-glucosidase yielded glucose
1
as the sugar moiety, as determined by co-HPTLC. The H NMR
data (Table 1) revealed signals for an olefinic methyl [δ 2.17 (3H,
d, J ) 1.3 Hz, H-10)], a methoxy [δ 3.66 (3H, s, H-11)], an
exocyclic olefinic methylene [δ 5.39 (1H, s, H-8ꢀ), 5.45 (1H, s,
H-8R)], a nonequivalent methylene [δ 2.73 (1H, br d, J ) 11.7
Hz, H-5R), 3.36 (1H, dd, J ) 11.7, 3.4 Hz, H-5ꢀ)], an olefinic
methine [δ 5.88 (1H, br s, H-2)], and two methines [δ 5.32 (1H, s,
H-9), 3.61(1H, d, J ) 3.4 Hz, H-4)], together with one glucose
unit [δ 5.53 (1H, d, J ) 7.8 Hz, H-1′), 3.60 (1H, m, H-2′), 3.51
(1H, m, H-3′), 3.45 (1H, m, H-4′), 3.52 (1H, m, H-5′), 3.70 and
3.92 (2H, m, H-6′)]. The 13C NMR data (Table 2) contained signals
for one olefinic methyl, one methoxy, two methylenes (one olefinic),
three methines (one olefinic, one acetal), and four quaternary
carbons (one ketone, two olefinic, one semiketal), together with
one glucose unit. This suggested that 3 is a monoterpene glucoside
with a structure similar to 1. However, when compared with 1,
compound 3 exhibited an exocyclic olefinic methylene and an
additional hemiacetal methine instead of a trisubstituted double bond
and an oxygenated methylene, which suggested the structure shown.
The HMBC correlations (Figure 1) of the glucosyl H-1′ with C-6,
the methoxy group proton with C-9, H-8 with C-4 and C-9, and
H-10 with C-2 confirmed the proposed structure for 3. Furthermore,
the ꢀ-glucosyl linkage was deduced from the coupling constant (J
) 7.8 Hz) of the anomeric proton at δ 5.53. The ROESY spectrum
showed correlations of H-1′ with H-5R, and H-4 with H-5ꢀ and
H-5R, and indicated that compound 3 has the same stereostructure
as compound 1. Thus, the structure of paeonin C was elucidated
as 3.
Figure 2. Key ROESY correlations of compounds 1 and 4.
resembled partly those of paeonisuffral and paeoniflorigenone,
isolated from Paeonia suffruticosa and Paeonia albiflora, respec-
tively.8,9
The HMBC correlations (Figure 1) of H-2 with C-1, C-4, and
C-10, H-9 with C-4 and C-6, and H-4 with C-3 confirmed the
structure for 1. Additionally, the HMBC correlation of H-1′ with
C-6 and H3-11 with C-8 suggested glucosylation occurred at C-6
and methoxylation at C-8. The ꢀ-glucosyl linkage was deduced
from the coupling constant of the anomeric proton at δ 5.43 (J )
7.9 Hz). The stereostructure of compound 1 was determined by
ROESY correlations of H-1′ with H-5R, H-4 with H-5R and H-5ꢀ,
and H-2 with Me-10 (Figure 2). Finally, the assignments of all
protons and carbons were made unambiguously by 1H-1H COSY,
HMQC, HMBC, and ROESY NMR experiments. On the basis of
the above evidence, the structure of paeonin A was determined
as 1.
Compound 4 was obtained as a colorless oil and gave the
molecular formula C17H26O10, on the basis of the HRESIMS (m/z
1
413.1398, [M + Na]+). The H and 13C NMR spectra (Tables 1
and 2) of 4 demonstrated the presence of a ketone carbonyl group,
a tertiary methyl group, a methoxy, an oxygenated methylene, two
methylenes, one acetal methine, and three quaternary carbons (two
oxygenated), besides characteristic glucose signals. Detailed analysis
of the NMR data revealed that the signals of 4 were very similar
to those of paeonidanin, a monoterpene glucoside isolated from
Paeonia peregrina and Paeonia lactiflora, except for the absence
of a benzoyl group linked with the OH at C-8.10,11 The HMBC
spectrum (Figure 1) revealed correlations of the glucosyl H-1′ with
C-1, H3-10 with C-1, H2-8 with C-1, H2-8 with C-9, H-9 with C-6,
Paeonin B (2), an amorphous powder, showed a molecular
formula of C16H22O9 by HRESIMS (m/z 381.1182, [M + Na]+).
1
The H and 13C NMR data (Tables 1 and 2) were very similar to
those of 1 except for the absence of a methoxy group. On the basis
1
of analysis of the HMBC (Figure 1), HMQC, and H-1H COSY
spectra, the structure of paeonin B was established as 2.

Notes
Journal of Natural Products, 2009, Vol. 72, No. 7 1323
Table 1. 1H NMR Data of Compounds 1-3 (400 MHz, J in Hz, pyridine-d5)
position
1
2
3
4
1
2
6.05 q (1.3)
6.04 q (1.3)
5.88 br s
3
2.51 br s
4
3.29 dd (3.4, 2.6)
2.48 dd (13.2, 3.4)
2.59 dd (13.2, 2.6)
3.27 dd (3.4, 2.6)
2.48 dd (13.1, 3.4)
2.56 dd (13.1, 2.6)
3.61 d (3.4)
2.73 br d (11.7)
3.36 dd (11.7, 3.4)
5R
5ꢀ
6
3.03 d (7.5)
7R
7ꢀ
8R
8ꢀ
9
2.21 d (11.0)
2.80 dd (11.0, 7.5)
3.60 s
3.69 d (11.9)
3.94 d (11.9)
6.70 s
3.65 d (11.9)
3.90 d (11.9)
6.78 s
5.45 s
5.39 s
5.32 s
5.04 s
10
11
1′
2′
3′
4′
5′
6′
2.06 d (1.3)
3.14 s
5.43 d (7.9)
3.68 m
3.57 m
3.43 m
3.46 m
3.73 m
3.94 m
2.04 d (1.3)
2.17 d (1.3)
3.66 s
1.60 s
3.36 s
5.39 d (7.9)
3.65 m
3.59 m
3.48 m
3.41 m
5.53 d (7.8)
3.60 m
3.51 m
3.45 m
3.52 m
3.70 m
3.92 m
5.16 d (7.4)
3.59 m
3.47 m
3.42 m
3.48 m
3.69 m
3.91 m
3.81 dd (12.0, 6.0)
3.99 dd (12.0, 6.0)
(Agilent 1100 series) was performed on a Zorbax Extend-C18 (Agilent
Technologies) column (4.6 × 250 mm; mobile phase, MeOH-H2O,
gradient elution; flow rate, 1.0 mL/min; detection, UV absorption at
210 and 254 nm). Column chromatographic separations were carried
out on silica gel H-60 (Qingdao Haiyang Chemical Group Corporation,
Qingdao, People’s Republic of China), Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden), and LiChroprep RP-18 (40-63 µm,
Merck). HSGF254 silica gel TLC plates (Yantai Chemical Industrial
Institute, Yantai, People’s Republic of China) were used for analytical
TLC. HPTLC plates (Merck) were used for co-TLC assay. D-
Glucopyranose (Chemprosa Holding AG) was used as a standard.
Molecular energy-minimized 3D models were calculated by Chemoffice
2004 software.
Plant Material. The roots of Paeonia lactiflora were supplied by
Shanghai LG Life Company, Shanghai, People’s Republic of China,
in July 2004. The materials were authenticated by Mr. Liang Wang of
Shanghai LG Life Company, where a specimen (No. 040510) is
deposited.
Extraction and Isolation. The air-dried and powdered roots of
Paeonia lactiflora (5.0 kg) were extracted exhaustively with 95% EtOH
(10 L × 3) at room temperature. The extract was concentrated in vacuo
to yield an EtOH extract (300 g), which was then suspended in distilled
water and partitioned successively with petroleum ether, EtOAc, and
n-BuOH. The EtOAc extract was concentrated in vacuo to give a residue
(65 g), which was applied to a silica gel column and eluted with a
gradient chloroform-methanol solvent system of increasing polarity
(50:1 f 10:1) to afford five fractions (A-E). Fraction B was subjected
and H3-11 with C-9. The ROESY spectrum (Figure 2) showed
correlations of H-1′ with H-7ꢀ, H-8 with H-9, H-5 with H-7R, and
H-10 with H-1′, H-3, and H-7R and suggested compound 4 has
the same relative stereostructure as paeonidanin.10,11 Further
analysis of the HMQC, HMBC, 1H-1H COSY, and ROESY spectra
confirmed the structure of compound 4 as 8-debenzoylpaeonidanin.
The monoterpene glucosides 1-3 possess new structural features
with double bonds and hemiketal units in a “cage-like” skeleton,
not found previously from Paeonia and other species. The yields
of 1-3 in the roots were very low. To confirm their natural
occurrence, the EtOAc fraction of the ethanol extract of P. lactiflora
was analyzed by HPLC and resulted in the detection of 1 and 3.
From the viewpoint of biosynthesis, it can be inferred that 1-3
are derived from paeonisuffral,8 a known monoterpene glucoside
from Paeonia suffruticosa, by cyclic cleavage, dehydration, meth-
ylation, and glucosylation. In a cell protection assay on compounds
1-4, compound 4 showed moderate protective activities against
H2O2-induced cell damage in PC12 cells (Figure 3).
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a Horiba Sepa-300 polarimeter. IR spectra were recorded using a
Perkin-Elmer 577 spectrometer. NMR spectra were run on a Bruker
AM-400 spectrometer with TMS as internal standard. LRESIMS were
measured using a Finnigan LCQ-DECA instrument, and HRESIMS
data were obtained on a JEOL SX-102 mass spectrometer. HPLC
Table 2. 13C NMR Data of Compounds 1-4 (100 MHz,
pyridine-d5)
position
1
2
3
4
1
2
3
4
5
6
7
8
154.7
127.5
195.9
44.0
32.2
98.6
108.9
70.5
142.9
17.2
57.1
97.8
74.9
78.4
71.1
78.6
62.5
154.8
127.5
196.2
44.0
32.4
98.4
112.0
60.3
141.1
17.1
161.1
124.3
196.4
47.9
33.8
97.6
138.3
117.2
102.8
17.1
55.8
97.6
87.9
85.6
48.5
204.6
46.7
62.9
26.0
59.1
105.8
20.3
55.8
96.3
74.9
78.3
70.2
79.0
62.1
9
10
11
1′
2′
3′
4′
5′
6′
98.4
74.8
78.4
71.1
78.7
62.4
75.0
78.7
71.6
79.1
Figure 3. Effects of compound 4 on cell viability. Viability of PC12
cells was obtained by measuring the MTT reduction. Two inde-
pendent experiments were carried out in triplicate. All data were
expressed as percentage of control values. ##p < 0.01 vs control.
**p < 0.01 vs H2O2 group.
62.8

1324 Journal of Natural Products, 2009, Vol. 72, No. 7
Notes
to repeated column chromatography over silica gel (CHCl3-MeOH,
30:1; 10:1) and LiChroprep RP-18 eluted with MeOH-H2O (3:2) to
afford compounds 1 (12 mg, 0.00024%) and 2 (8 mg, 0.00016%).
Fraction D was separated by repeated column chromatography over
silica gel (CHCl3-MeOH, 10:1; 5:1) and LiChroprep RP-18 eluted with
MeOH-H2O (1:1) and Sephadex LH-20 eluted with MeOH to afford
compounds 3 (13 mg, 0.00025%) and 4 (5 mg, 0.00010%).
monitoring of UV absorption at 210 and 254 nm. The mobile phase
used was a mixture of MeOH and H2O in which the proportion of
MeOH was increased from 10% to 100% over a 60 min period. The
flow rate was 1.0 mL/min. This resulted in the detection of 1 and 3 in
the EtOAc extract. The retention times of compounds 1-4 were 26.16,
19.21, 17.51, and 16.35 min, respectively.
Assay for Cell-Protective Activities. Evaluation of cell-protective
activities of compounds 1-4 against H2O2-induced PC12 cell damage
was performed according to a protocol reported in the literature.12 Cells
were incubated with 200 µM H2O2 for 30 min, and the cultures were
further developed for another 6 h. Samples were added to the cultures
2 h prior to H2O2 addition. Cell viability was obtained by measuring
MTT reduction. Two independent experiments were carried out in
triplicate. All data were expressed as a percentage of control values.
Statistical comparison was made by using one-way ANOVA and
followed by Duncan’s test. The data were expressed as means ( SEM.
Paeonin A (1): colorless oil; [R]20 -36.5 (c 0.1, pyridine); UV
D
(MeOH) λmax (log ꢀ) 252 (3.84) nm; IR (KBr) νmax 3365, 1679, 1640,
1612 cm-1; 1H NMR (400 MHz, pyridine-d5) and 13C NMR data (100
MHz, pyridine-d5), see Tables 1 and 2; HRESIMS m/z [M + Na]+
395.1286 (calcd for C17H24O9Na, 395.1318).
Paeonin B (2): white, amorphous powder; [R]20 -37.8 (c 0.1,
D
pyridine); UV (MeOH) λmax (log ꢀ) 254 (3.82) nm; IR (KBr) νmax 3360,
1675, 1638, 1610 cm-1; 1H NMR (400 MHz, pyridine-d5) and 13C NMR
data (100 MHz, pyridine-d5), see Tables 1 and 2; HRESIMS m/z [M
+ Na]+ 381.1182 (calcd for C16H22O9 Na, 381.1161).
References and Notes
Paeonin C (3): white, amorphous powder; [R]20 -45.1 (c 0.1,
D
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cao; Shanghai Science and Technology Publishing Company: Shang-
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Yoshikawa, M. Chem. Pharm. Bull. 1996, 44, 1279–1281.
(5) Takagi, K.; Harada, M. Yakugaku Zasshi 1969, 89, 887–890.
(6) Hsu, F. L.; Lai, C. W.; Cheng, J. T. Planta Med. 1997, 63, 323–325.
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pyridine); UV (MeOH) λmax (log ꢀ) 256 (3.80) nm; IR (KBr) νmax 3370,
1668, 1650, 1613 cm-1; 1H NMR (400 MHz, pyridine-d5) and 13C NMR
(100 MHz, pyridine-d5), see Tables 1 and 2; HRESIMS m/z [M + Na]+
395.1326 (calcd for C17H24O9Na, 395.1318).
8-Debenzoylpaeonidanin (4): colorless oil; [R]20 -30.2 (c 0.1,
D
pyridine); IR (KBr) νmax 3430, 1700 cm-1
;
1H NMR (400 MHz,
pyridine-d5) and 13C NMR (100 MHz, pyridine-d5), see Tables 1 and
2; HRESIMS m/z [M + Na]+ 413.1398 (calcd for C17H26O10Na,
413.1424).
Hydrolysis of Compounds 1 and 3. Compound 1 (4 mg) was
dissolved in H2O (8 mL) and incubated with almond ꢀ-glucosidase
(20 mg) for 24 h at 37 °C. After general treatment, the sugar moiety
was analyzed by silica gel HPTLC (Merck) developed with Me2CO-2
mM NaOAc (17:3, v/v) and detected by spraying with 0.2% naph-
thoresorcinol in Me2CO-3 N H3PO4 (5:1, v/v), followed by heating at
105 °C for 5 min (Rf 0.53). D-Glucopyranose was used as standard.
The same procedure was used for compound 3.
HPLC Analysis of the EtOAc Extract of P. lactiflora. The EtOAc
extract and compounds 1-4 were analyzed by HPLC using a Zorbax
Extend-C18 (Agilent Technologies) column (4.6 × 250 mm) with
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NP8001783