Monoterpene derivatives with anti-allergic activity from red peony root, the root of Paeonia lactiflora

Article abstract of DOI:10.1016/j.fitote.2015.11.011

The methanolic extract and its subfractions from red peony root, the dried roots of Paeonia lactiflora Pallas showed potent antiallergic effects, as inhibition of immunoglobulin E (IgE)-mediated degranulation in rat basophil leukemia (RBL)-2H3 cells. Bioassay-guided fractionation led to the isolation of 16 monoterpene derivatives, including 3 new compounds, paeoniflorol (1), 4′-hydroxypaeoniflorigenone (2) and 4-epi-albiflorin (3), together with 13 known ones (4-16). The chemical structures of the new compounds were elucidated on the basis of spectroscopic and chemical evidences. Among the isolated monoterpene derivatives, nine compounds showed potent anti-allergic effects and compound 1 was the most effective. A primary structure-activity relationship of monoterpene derivatives was discussed.

Full text of DOI:10.1016/j.fitote.2015.11.011

Fitoterapia 108 (2016) 55–61
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Monoterpene derivatives with anti-allergic activity from red peony root,
the root of Paeonia lactiflora
a
a
b
c
Yan-Hong Shi a, Shu Zhu a, , Yue-Wei Ge , Yu-Min He , Kohei Kazuma , Zhengtao Wang ,
⁎
Kayo Yoshimatsu d, Katsuko Komatsu a,
⁎
a
Division of Pharmacognosy, Department of Medicinal Resources, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan
Division of Kampo-Pharmaceutics, Department of Medicinal Resources, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan
The MOE Key laboratory of Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201-203, China
Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki 305-0843, Japan
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
The methanolic extract and its subfractions from red peony root, the dried roots of Paeonia lactiflora Pallas
showed potent antiallergic effects, as inhibition of immunoglobulin E (IgE)-mediated degranulation in rat baso-
phil leukemia (RBL)-2H3 cells. Bioassay-guided fractionation led to the isolation of 16 monoterpene derivatives,
including 3 new compounds, paeoniflorol (1), 4′-hydroxypaeoniflorigenone (2) and 4-epi-albiflorin (3), together
with 13 known ones (4–16). The chemical structures of the new compounds were elucidated on the basis of
spectroscopic and chemical evidences. Among the isolated monoterpene derivatives, nine compounds showed
potent anti-allergic effects and compound 1 was the most effective. A primary structure-activity relationship of
monoterpene derivatives was discussed.
Received 9 September 2015
Received in revised form 13 November 2015
Accepted 14 November 2015
Available online 18 November 2015
Keywords:
Red peony root
Paeonia lactiflora
Monoterpene derivatives
Anti-allergic activity
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
after initial stimulation with the antigen, and then signal sensitized
mast cells and basophils via high affinity IgE receptor (FcεRI) to release
Peony root, called “Shaoyao” in Chinese and “Shakuyaku” in
Japanese, is one of widely-used crude drugs in traditional Chinese med-
icine and Kampo medicine [1–2]. In China, there are two kinds of peony
root, white peony root (WPR) and red peony root (RPR) which are used
for different therapeutic purposes, however, most of them are derived
from the same botanical origin Paeonia lactiflora Pallas [3]. We have
previously demonstrated that WPR and RPR, although derived from
the same P. lactiflora, are genetically and chemically different and
geographically isolated from each other [4]. Monoterpene compounds
with a “cage-like” pinane skeleton are considered as the main and
characteristic bioactive constituents of peony root, which have been
revealed to have anti-inflammatory, anti-diabetic, anti-oxidant,
anti-allergic, anti-coagulative, sedative and analgesic activities, etc.
[5–8]. Recently, several studies reported anti-allergic activity of peony
root [9–11], but detailed investigation of chemical constituents with
anti-allergic activity, especially these characteristic monoterpenoids,
has not been done. Type I allergic reactions are known to be triggered
by binding of antigen to specific IgE antibody which has been produced
allergic mediators, such as β-hexosaminidase, histamine and cytokines
[10,12–13]. Therefore, β-hexosaminidase is considered as a degranula-
tion marker and inhibition of β-hexosaminidase release in rat basophil
leukemia (RBL)-2H3 cells has been widely employed as an in vitro
model of IgE-mediated degranulation in mast cells for screening
candidates with anti-allergic drugs from natural resources [14–16]. In
a bioactivity screening experiment, RPR produced in Inner Mongolia
autonomous region of China showed the relatively strong anti-allergic
activity among 80 crude drugs registered in Japanese Pharmacopeia,
rather than the WPR [17]. Therefore, subsequent bioassay-guided
fractionation of the methanol extract of RPR was conducted, which
led to isolation of 16 monoterpene derivatives, including 3 new
compounds, and their anti-allergic activity was further investigated.
2. Experimental
2.1. General experimental procedures
NMR spectra were recorded on a JEOL ECX-400 spectrometer (1H,
400 MHZ; 13C, 100 MHZ) with TMS as an internal standard. ESI-MS
spectra were measured by Agilent HPLC system with an ion-trap mass
spectrometer fitted with an electrospray ionization source (Bruker
Daltonics Inc., Germany). HR-ESI-MS spectra were measured by Accella
HPLC system equipped with an Orbitrap-EX mass spectrometer (Thermo
⁎
Corresponding authors at: Division of Pharmacognosy, Department of Medicinal
Resources, Institute of Natural Medicine, University of Toyama, Sugitani 2630, Toyama
930-0194, Japan.
E-mail addresses: szhu@inm.u-toyama.ac.jp (S. Zhu), katsukok@inm.u-toyama.ac.jp
(K. Komatsu).
http://dx.doi.org/10.1016/j.fitote.2015.11.011
0367-326X/© 2015 Elsevier B.V. All rights reserved.

56
Y.-H. Shi et al. / Fitoterapia 108 (2016) 55–61
Fisher Scientific, Inc., USA). IR spectra were measured by using a JASCO
FT/IR-460 Plus spectrophotometer (JASCO International Co. Ltd., Japan).
Optical rotations were detected by using a JASCO P2100 digital polarim-
eter (JASCO International Co. Ltd., Japan). Column chromatography (CC)
was performed with normal-phase (Wakogel® C-200 and 300HG, Wako
Pure Chemical Industries, Ltd., Japan), reverse-phase [ODS-A (12 nm, s-
150 μm) and ODS-AQ-HG (12 nm, s-50 μm), YMC Co., Ltd., Japan], and
Sephadex LH-20 (GE Healthcare Life Sciences, Sweden). Preparative
HPLC was performed on a Waters HPLC system (Waters Co. Ltd., USA)
equipped with a Delta 600 pump and 2489 UV detector by using
YMC-Pack ODS-A column (5 μm, 250 × 20 mm i.d.). TLC was carried
and 5 (33.0 mg) from 80MF-7-3, and 2 (2.0 mg) from 80MF-7-5
were also achieved on the preparative HPLC eluted with MeCN–H2O
(20:80, v/v).
2.3.1. Paeoniflorol (1)
White, amorphous powder; [α]2D0 −33.33° (c = 0.13, MeOH); IR
(KBr): νmax 3418, 1715, 1631, 1456, 1281, 1075, 712 cm−1 1H– and
;
13C NMR data are showed in Table 1; ESI-MS: m/z 511 [M + HCOO]−
;
,
HR-ESI-MS: m/z 511.1812 [M + HCOO]− (calcd. for C24H31O12
511.1816).
out to monitor fractions on precoated silica gel (60F254) or RP-18 (F254
)
2.3.2. 4′-hydroxypaeoniflorigenone (2)
plates (0.50 mm thickness, Merck, Germany). Spots were visualized
under UV (254 and 360 nm) and/or monitored by spraying with the
solution of 10% sulfuric acid in ethanol (v/v). L-cysteine methyl ester
hydrochloride, phenyl isothiocyanate, L-(−)-glucose (purity: ≥98%),
White, amorphous powder; [α]2D0 +4.99° (c = 0.10, MeOH); IR
(KBr): νmax 3402, 1712, 1609, 1514 cm−1 1H– and 13C NMR data are
;
given in Table 1; ESI-MS: m/z 335 [M + H]+; HR-ESI-MS: m/z
335.1124 [M + H]+ (calcd. for C17H19O7, 335.1124).
D-(+)-glucose (purity: ≥98%), and pyridine were purchased from Wako
Pure Chemical Industries, Ltd.
2.3.3. 4-epi-albiflorin (3)
White, amorphous powder; [α]2D0 −21.40° (c = 0.11, MeOH); IR
(KBr): νmax 3389, 1755, 1715, 1631, 1452, 1280, 1075, 715 cm−1; 1H–
and 13C NMR data are given in Table 1; ESI-MS: m/z 525
[M + HCOO]−; HR-ESI-MS: m/z 525.1617 [M + HCOO]− (calcd. for
2.2. Plant material
Red peony root was purchased from Chifeng Rongxintang Pharma-
ceutical Co. Ltd., Inner Mongolia autonomous region, China, in Oct.
2013, which was clearly identified as RPR type of P. lactiflora by genetic
analysis of nrDNA ITS sequence [4]. The voucher (TMPW No. 27967) is
deposited in the Museum of Materia Medica, Institute of Natural
Medicine (TMPW), University of Toyama, Japan.
C24H29O13, 525.1608).
2.4. Acid hydrolysis and sugar analysis of compounds 1 and 3
Acid hydrolysis of 1 and 3 was carried out using a procedure similar
to previous report [15]. Briefly, compounds 1 and 3 (1.0 mg, respective-
ly) were hydrolyzed by heating in 1.0 M HCl (0.2 ml) at 90 °C for 2 h and
then neutralized with Amberlite IRA400. After drying in vacuo, the res-
idue was dissolved in pyridine (0.1 ml) containing L-cysteine methyl
ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h, and then
0.1 ml solution of phenyl isothiocyanate (0.5 mg) was added to the
mixture and heated at 60 °C for 1 h. The reaction solution was directly
analyzed by HPLC-PDA system to determine the D/L configuration of
sugar moieties by comparing their retention time (tR) with the
authentic D- and L-glucose derivatives (tR: D-glucose, 18.03 min; L-
glucose, 17.02 min).
2.3. Extraction and isolation
The dried roots (1.5 kg) were cut into small pieces and then extract-
ed repeatedly with methanol (3 × 2.5 L) by ultrasonic for 2 h at room
temperature. The MeOH-extract (280.0 g) was obtained after removing
the solvent under vacuum, and then was subjected to Daion HP 21 CC
eluted with a gradient of H2O and MeOH as 0, 20%, 40%, 60%, 80% and
100% MeOH (v/v), respectively. The anti-allergic activity assay showed
that the 60% and 80% aqueous MeOH-elutions had significant inhibitory
activities against β-hexosaminidase release in IgE-sensitized RBL-2H3
cells, so the two elutions were further isolated for tracking the active
constituents. The 60% MeOH-elution (30.8 g) was suspended in water
and partitioned with EtOAc and n-BuOH, respectively. The n-BuOH-
soluble fraction was then subjected to YMC-ODS-A CC eluted with
MeOH–H2O (1:9 to 1:0, v/v) to obtain six fractions (60MBF-1 to 6).
The 60MBF-2 was further subjected to YMC-ODS AQ CC eluted with
MeOH–H2O (1:9 to 1:0, v/v) to give seven fractions (60MBF-2-1 to 7).
60MBF-2-3 was separated by preparative HPLC eluted with isocratic
acetonitrile(MeCN)-H2O (15:85, v/v) to give 1 (6.0 mg) and 8
(5.0 mg). Compound 11 (20.0 mg) was obtained from 60MBF-2-4 by
using sephadex LH-20 CC with MeOH–H2O (6:4, v/v). The 60MBF-3
was separated by YMC-ODS-A CC eluted with MeOH–H2O (1:1, v/v) to
give four fractions (60MBF-3-1 to 4). Compounds 7 (5.0 mg) and 12
(3.0 mg) were purified from 60MBF-3-1 by using preparative HPLC
with isocratic elution of MeCN–H2O (15:85, v/v). The 60MBF-3-2 was
further separated by sephadex LH-20 CC eluted with MeOH–H2O (6:4,
v/v) to give 15 (2.0 mg). In addition, 80% MeOH-elution (5.9 g) was sub-
jected to a normal phase silica gel CC eluted by CH3Cl–MeOH with gra-
dient condition from 1:0 to 1:5 (v/v) to give seven fractions (80MF-1 to
7). The 80MF-1 was purified by the preparative HPLC with MeCN–H2O
(30:70, v/v) to give 4 (10.0 mg) and 6 (18.0 mg). Compounds 9
(100.0 mg) and 16 (10.0 mg) were purified from 80MF-3 under the
same pre-HPLC condition. The 80MF-7 was further separated by
sephadex LH-20 CC with MeOH–H2O (6:4, v/v) to give five subfractions
(80MF-7-1 to 5). The isolation of 14 (33.0 mg) from 80MF-7-2, 10
(50.0 mg) and 13 (40.0 mg) from 80MF-7-4 were achieved by using pre-
parative HPLC with MeCN–H2O (30:70, v/v). The isolation of 3 (8.0 mg)
2.5. Inhibitory effects on the release of β-hexosaminidase from RBL-2H3
cells
The inhibitory effects of the test samples against 2, 4-
dinitrophenylated bovine serum albumin (DNP-BSA) stimulated β-
hexosaminidase release in IgE-sensitized RBL-2H3 cells (Cell No.
RCB2782, RIKEN Cell Bank, Tsukuba, Japan) were evaluated by a previ-
ously reported method [16,18]. Briefly, RBL-2H3 cells were dispended
into 96-well culture plates at a concentration of 4 × 105 cells/well
using Eagle's Minimum Essential Medium (MEM, Gibco, Thermo Fisher
Scientific Inc., USA) supplementing with 10% Fetal Bovine Serum (FBS,
12003C, Sigma-Aldrich Co. LLC., USA), penicillin (100 U/ml, Sigma-
Aldrich) and streptomycin (100 μg/ml, Sigma-Aldrich) and cultured
overnight at 37 °C in 5% CO2. After washing with phosphate-buffered sa-
line (PBS, Gibco), the cells were sensitized with 0.5 μg/ml of anti-DNP
IgE (D8406, Sigma-Aldrich) for 24 h, and then washed twice with
200 μl Siraganian buffer [119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2,
25 mM piperazine-N,N’-bis (2-ethanesulfonic acid) (PIPES), 40 mM
NaOH; pH 7.2] and incubated in 160 μl Siraganian (+) buffer [Added
5.6 mM glucose, 1 mM CaCl2 and 0.1% bovine serum albumin (BSA,
A7030, Sigma-Aldrich Co. LLC., USA) in Siraganian buffer; pH 7.2] for
10 min at 37 °C. Subsequently, 50 μl of test sample solution was added
to each well and incubated for 30 min, followed by adding 50 μl DNP-
BSA (1.0 μg/ml, A23018, Thermo Fisher Scientific Inc.) as an antigen
for 1 h to stimulate cells and to evoke allergic reactions (degranulation).
The reaction was stopped by cooling in an ice-bath for 10 min, and then
100 μl of the supernatant was transferred to a new 96-well plate and

Y.-H. Shi et al. / Fitoterapia 108 (2016) 55–61
57
Table 1
13C NMR (100 MHz) and 1H NMR (400 MHz) spectroscopic data for 1, 2, 3 and 4 measured in CD3OD.
Position
1
2
3
4
δ C
δ H (J in Hz)
δ C
δ H (J in Hz)
δ C
δ H (J in Hz)
δ C
δ H (J in Hz)
1
2
87.9
91.3
80.0
48.1
86.1
92.4
86.9
93.5
2.53, d (16.0); 2.77,
d (16.0)
3
45.5
1.84, d (16.0); 2.41,
dd (8.0, 16.0)
212.5
42.3
2.03, dd (8.0, 14.0); 2.39,
d (14.0)
41.7
2.01, dd (1.6, 15.6); 2.40,
dd (6.8, 15.6)
4
5
69.9
40.5
4.17, dd (4.0, 8.0)
2.59, d (4.0)
47.9
35.6
2.88, d (7.6)
2.28, dd (6.8, 13.2); 2.37,
dd (5.2, 10.0)
66.7
40.3
4.03, t (7.2)
2.77, d (7.2)
68.4
41.6
4.26, dd (4.8, 6.4)
2.91, dd (5.2, 7.6)
6
7
59.0
28.2
102.8
44.5
58.7
25.8
56.9
28.5
1.73, d (9.6); 2.60, d (1.2)
4.65, d (12.0); 4.73, d (12.0)
2.33, d (7.2)
2.35, dd (5.2, 12.0); 2.72,
dd (7.2, 12.0)
4.69, d (12.0); 4.83, d (12.0)
2.04, d (11.2); 2.79,
dd (8.0, 11.2)
4.67, d (12.4); 4.80,
d (12.4)
8
67.3
63.7
3.99, dd (8.8, 11.6); 4.20,
dd (6.0, 11.6)
5.39, s
61.3
62.0
9
10
70.5
21.7
3.77, d (8.0); 4.78, d (8.0)
1.33, s
101.0
21.8
176.5
20.2
178.0
20.5
1.24, s
1.53, s
1.52, s
1-O-glucose
1′
2′
3′
4′
5′
6′
100.1
75.1
77.9
71.8
78.1
63.0
4.49, d (7.6)
3.23, m
3.37, m
3.29, m
3.31, m
100.1
74.8
78.0
71.6
78.1
62.7
4.54, d (8.0)
3.21, m
3.23, m
3.20, m
3.32, m
100.1
74.8
78.0
71.6
78.2
62.8
4.52, d (8.0)
3.21, m
3.24, m
3.22, m
3.30, m
3.61, dd (4.8, 11.6); 3.84,
dd (0.8, 11.6)
3.62, dd (4.8, 11.6); 3.85,
d (11.6)
3.62, dd (4.4, 11.6);
3.84, d (11.6)
8-O-benzoyl
1″a
2″a
3″a
4″a
5″a
6″a
7″a
131.4
130.7
129.7
134.4
129.7
130.7
168.2
122.0
132.8
116.2
163.7
116.2
132.8
167.7
131.1
130.7
129.7
134.5
129.7
130.7
167.8
131.3
130.8
129.7
134.4
129.7
130.8
167.9
8.04, dd (1.6, 6.8)
7.48, dd (1.6, 7.6)
7.60, m
7.48, dd (1.6, 7.6)
8.04, dd (1.6, 6.8)
7.86, dt (2.8, 8.4)
6.81, dt (2.8, 8.4)
8.04, dd (1.2, 8.0)
7.49, t (8.0)
7.60, m
7.49, t (8.0)
8.04, dd (1.2, 8.0)
8.07, dd (1.2, 8.0)
7.49, t (8.0)
7.62, m
7.49, t (8.0)
8.07, dd (1.2, 8.0)
6.81, dt (2.8, 8.4)
7.86, dt (2.8, 8.4)
a
Position no. is 1′ to 7′ in compound 2.
incubated with 50 μl of substrate (3.3 mM p-nitrophenyl-N-acetyl-β-D-
glucosaminide) for 1 h at 37 °C. The reaction was stopped by adding
100 μl of alkaline buffer (0.1 M NaHCO3 and 0.1 M Na2CO3; pH 10)
and cooled in an ice-bath for 10 min. Then the absorbance at 405 nm
was measured using microplate reader. The test samples were dissolved
in dimethylsulfoxide (DMSO) and Siraganian (+) buffer (final DMSO
concentration was less than 0.1%).
The percentage of cell viability was calculated according to the follow-
ing formula: Cell viability (%) = (Mean absorbance of test samples/
Mean absorbance of vehicle control) × 100.
3. Results and discussion
Methanolic extract of red peony root showed potent anti-allergic
activity as inhibitory effects against DNP-BSA stimulated β-
hexosaminidase release in IgE-sensitized RBL-2H3 cells (IC50: 0.61
mg/ml). After fractionation with Daion HP-21 CC eluted by a series
of aqueous MeOH solutions, 60% and 80% aqueous MeOH fractions
were found to exhibit significantly inhibitory effects on β-
hexosaminidase release (IC50: 0.20 and 0.12 mg/ml, respectively).
Therefore, the 60% and 80% aqueous MeOH fractions were subjected
to normal- and reversed-phase CC followed by preparative HPLC to
afford three new monoterpene derivatives, paeoniflorol (1), 4′-
hydroxypaeoniflorigenone (2), 4-epi-albiflorin (3), and 13 known
monoterpene glycosides (4–16) (Fig. 1). The spectral data of
compounds 4–16 were consistent with those reported in previous
literatures, therefore were identified as albiflorin (4) [19], paeonivayin
(5) [20], paeoniflorin (6) [19], 4-O-methyl-paeoniflorin (7) [21],
salicylpaeoniflorin (8) [22], benzoylpaeoniflorin (9) [19], moudanpioside
C (10) [23], galloylpaeoniflorin (11) [24], oxypaeoniflorin (12) [19],
benzoyloxypaeoniflorin (13) [23], 6′-O-vanillyloxypaeoniflorin (14)
[25], paeonidanin E (15) [5], and lactiflorin (16) [19]. The structures of
the 3 new compounds were determined as follows.
The inhibitory activity against β-hexosaminidase release of the test
samples was calculated using the following equation:
Inhibition ð%Þ ¼ ½1−ðAt−Ab−AnÞ=ðAc−AnÞꢀ ꢁ 100
At
test: DNP-BSA (+), sample (+)
normal: DNP-BSA (−), sample (−)
blank: DNP-BSA (−), sample (+)
An
Ab
Ac
control, DNP-BSA (+), sample (−).
The results were expressed as mean S.D. for three independent
experiments. One-way ANOVA followed by post-hoc Dunnett's test or
Chi-square test were used for statistical analysis, and IC50 values were
calculated by using SPSS software (version 15.0, IBM, USA).
2.6. Measurement of cell viability
The cytotoxicity of extract, fractions and isolated compounds on
RBL-2H3 cells was evaluated with Cell Counting kit-8 (Dojindo labora-
tories Co. Ltd., Japan) according to manufacturer's instructions. Briefly,
anti-DNP IgE-sensitized cells were treated with various sample
solutions and stimulated by DNP-BSA as described above. After wash
with PBS, 100 μl 10% FBS-MEM containing 10 μl WST-8 was added to
each well and incubated for 3 h. Then, the absorbance at 450 nm was
measured by using a microplate reader (Infinite® F200, Switzerland).
Compound
1 was isolated as white amorphous powder. A
quasimolecular ion at m/z 511.1812 [M + HCOO]− in the HR-ESI-MS
spectrum suggested a molecular formula of C23H30O10. The IR spectrum
of 1 showed absorption bands at 3418 cm−1 (hydroxyl group),
1715 cm−1 (carbonyl group), 1631 and 1456 cm−1 (aromatic ring).
The 1H NMR spectrum of 1 (Table 1) showed signals of a tertiary methyl

58
Y.-H. Shi et al. / Fitoterapia 108 (2016) 55–61
Fig. 1. Structures of monoterpene derivatives isolated from the roots of P. lactiflora.
at δH 1.33 (3 H, s, H-10), two methylenes at δH 1.84 (1 H, d, J = 16.0 Hz,
H-3β), 2.41 (1 H, dd, J = 8.0, 16.0 Hz, H-3α), and at δH 1.73 (1 H, d, J =
9.6 Hz, H-7α), 2.60 (1 H, d, J = 1.2 Hz, H-7β), together with two
oxymethylenes at δH 4.65, 4.73 (2 H, J = 12.0 Hz, H-8) and δH 3.77,
4.78 (2 H, J = 8.0 Hz, H-9). The 1H NMR of 1 also indicated a series of
proton signals assignable to a glucopyranosyl moiety and a benzoyl
moiety. In addition, the acid hydrolysis of 1 yielded β-D-glucopyranose.
The 13C NMR spectrum of 1 showed signals at δC 21.7 (C-10), 45.5 (C-3),
28.2 (C-7) 67.3 (C-8) and 70.5 (C-9), which were in good accordance
with those reported for 4α-hydroxyl derivative of paeonisuffrone
(PSOH) [26–27], except for the signals of C-1 and C-8. The deduced
monoterpene skeleton was also supported by the cross peaks from the
proton H-4 (δH 4.17) to H-3α (δH 2.41) and to H-5 (δH 2.59) in 1H–1H
COSY, and the cross peak from H-4 (δH 4.17) to C-4 (δC 69.9) in HMQC
spectrum. The downfield shift of C-1 (1: δC 87.9, PSOH: δC 82.6) and
the long-range correlation between H-1′ (δH 4.49) and C-1 in HMBC
spectrum indicated that glucosylation at C-1 of the monoterpene
skeleton. The downfield shift of C-8 (1: δC 67.3, PSOH: δC 64.9) and the
long-range correlation between H-8 (δH 4.65 and δH 4.73) and C-7″ in
HMBC spectra confirmed that the benzoyl moiety was connected to C-
8 (Fig. 2). The β-configuration of the hydrogen at C-4 of 1 was confirmed
by the NOESY cross signals between H-4 and H-3β, H-4 and H-7α, as
well as between H-3β and H-7α (Fig. 2). Consequently, compound 1
was identified as 8-benzoyl-1-O-β-D-glucopyranosyl-4-hydroxyl deriv-
ative of paeonisuffrone, and named as paeoniflorol.
group), 1609 and 1514 cm−1 (aromatic ring). The 1H NMR spectrum of
2 (Table 1) showed signals of a tertiary methyl at δH 1.24 (3 H, s, H-10),
an oxymethylene at δH 3.99 (dd, J = 8.8, 11.6 Hz, H-8α) and δH 4.20 (J =
6.0, 11.6 Hz, H-8β), an acetal at δH 5.39(1 H, s, H-9), and as well as four
aromatic protons at δH 6.81(2 H, dt, J = 2.8, 8.4 Hz, H-3″ and H-5″) and
δH 7.86 (2 H, dt, J = 2.8, 8.4 Hz, H-2″ and H-6″). The 13C NMR of 2
showed a set of diagnostic signals for a ketonic carbonyl (δC 212.5, C-
3), an ester carbonyl (δC 167.7, C-7′), a methyl (δC 21.8, C-10), three
methylenes (δC 35.6, C-5; δC 48.1, C-2; δC 63.7, C-8), three methines
(δC 44.5, C-7; δC 47.9, C-4; δC 101.0, C-9), two quaternary carbons (δC
80.0, C-1; δC 102.8, C-6), and six aromatic carbons (δC 116.2–163.7),
which closely resembled to those of paeoniflorigenone [27–28], except
for the aromatic carbons. Both the 1H and 13C NMR data indicated
clearly the presence of a p-hydroxybenzoyl moiety. In addition, the
correlation from H-8 to C-7′ in HMBC spectrum confirmed the
p-hydroxylbenzoyl moiety was connected to C-8 (Fig. 2). Therefore,
compound 2 was identified as 4′-hydroxypaeoniflorigenone.
Compound 3 was isolated as white amorphous powder. The HR-
ESI-MS of 3 displayed a quasimolecular ion peak at m/z 525.1617
[M + HCOO]−, which suggested a molecular formula of C23H28O11. The
IR spectrum of 3 indicated absorption bands at 3389 cm−1 (hydroxyl
group), 1755 and 1715 cm−1 (carbonyl group), 1631 and 1452 cm−1
(aromatic ring). The 1H NMR spectrum of 3 showed a methyl at δH 1.53
(3 H, s, H-10), two methylenes at δH 2.03 (1 H, dd, J = 8.0, 14.0 Hz, H-
3β), 2.39 (1 H, d, J = 14.0 Hz, H-3α) and at δH 2.35 (1 H, dd, J = 5.2,
12.0 Hz, H-7α), 2.72 (1 H, dd, J = 7.2, 12.0 Hz, H-7β), two methines at
Compound
2 was isolated as white amorphous powder. A
quasimolecular ion at m/z 335.1124 [M + H]+ in the HR-ESI-MS sug-
gested a molecular formula of C17H18O7. The IR spectrum of 2 indicated
absorption bands at 3402 cm−1 (hydroxyl group), 1712 cm−1 (carbonyl
δH 4.03 (1 H, t, J = 7.2 Hz, H-4) and at δH 2.77 (1 H, d, J = 7.2 Hz, H-5),
one oxymethylene at δH 4.69, 4.83 (2 H, J = 12.0 Hz, H-8), five aromatic
protons at δH 7.49 (2 H, t, J = 8.0 Hz, H-3″ and H-5″), δH 7.60 (1 H, m, H-

Y.-H. Shi et al. / Fitoterapia 108 (2016) 55–61
59
Fig. 2. Important 2D-NMR correlations of compounds 1, 2, 3 and 4.
4″) and δH 8.04 (2 H, dd, J = 1.2, 8.0 Hz, H-2″ and H-6″), together with a
set of proton signals assignable to a glucopyranosyl moiety (Table 1).
Correspondently, the 13C NMR spectrum displayed 23 carbon signals,
which included characteristic signals of a methyl (δC 20.2, C-10), two
methylenes (δC 42.3, C-3; δC 25.8, C-7), two methines (δC 66.7, C-4; δC
40.3, C-5), one oxymethylene (δC 61.3, C-8), two ester carbonyl (δC
176.5, C-9; δC 167.8, C-7″), six aromatic carbons (δC 131.1, C-1″; δC
130.7, C-2″, 6″; δC 129.7, C-3″, 5″; δC 134.5, C-4″), and six glucopyranosyl
carbons (δC 100.1, 78.1, 78.0, 74.8, 71.6, 62.7) (Table 1). The 1D NMR (1H
and 13C) and 2D NMR (HMQC, HMBC) data of 3 were closely similar to
those of albiflorin (4), which suggested 3 had the same planar structure
with 4 [19,29–30]. Carefully compared their NMR data, significant differ-
ences were found in the coupling constant of the methine proton H-4 (3:
J = 7.2 Hz; 4: J = 4.8, 6.4 Hz), as well as the chemical shifts of the meth-
ylene signals C-7 and H-7α (3: δC 25.8, δH 2.35; 4: δC 28.5, δH 2.04), and
the methine signals C-5 and H-5 (3: δC 40.3, δH 2.77; 4: δC 41.6, δH 2.91),
which suggested that 3 was an epimer of 4. The stereochemistry of hy-
droxyl group at C-4 in 4 was confirmed to be α-configuration [18]. In
the 1D different NOE spectrum of 4, correspondently, the correlations
from H-4 to H-3β and H-7α, as well as from H-3β to H-7α were ob-
served (Fig. 2). By contrast, the NOE correlation between H-4 (δH
4.03) and H-3α was observed in 3, suggesting the co-facial of these

60
Y.-H. Shi et al. / Fitoterapia 108 (2016) 55–61
two protons with α-orientation. Moreover, the presence of NOE corre-
lation between H-3β and H-7α on the opposite side and the absence
of correlation between H-4 and H-7α definitely indicated that the hy-
droxyl moiety at C-4 in 3 was β-configuration. Therefore, compound 3
was determined to be the epimer of albiflorin and named as 4-epi-
albiflorin.
galloyl group attaching to C-6″ of glucose moiety seemed to enhance
the activity (11 vs. 9–10).
Acknowledgments
This work was supported by JSPS KAKENHI Grant Numbers
24406005 and 15H05268, and by a grant from Japan Agency for Medical
Research and Development (15ak0101034h0001).
Monoterpenoids possessing a “cage-like” pinane skeleton, such as
paeoniflorin, albiflorin, and paeonisuffrone, have been only found in
the genus Paeonia, the single genus in Paeoniaceae. Up to dates, more
than 60 monoterpenoids have been isolated and identified from Paeonia
species. Although such unique monoterpenoids are presumed to be
formed initially by the cyclization of geranyl pyrophosphate, followed
by multi-steps of oxidation, cyclization, benzoylation, glycosylation,
etc., experimental evidences of biosynthetic pathway in detail are scanty,
and biogenetic relationship within various classics of monoterpenoids is
still unclear. In the present study, an acetone soaking extract of
P. lactiflora root was prepared and analyzed by LC-MS, and all of the
three new compounds were detected, which suggested that these com-
pounds are natural metabolites contained in this red peony root. From
the viewpoint of biosynthesis, the compound 1 is proposed to be derived
from paeonisuffrone [27] by enzymatic reduction with alcohol dehydro-
genase (ADH) which can catalyze the reduction of aldehydes and
ketones to alcohols [31–32]. The compound 2 can be derived from
paeonisuffral [27] through p-hydroxybenzoylation of C8-OH. The
compound 3 with β configuration of C4-OH is not isolated previously
from Paeonia species, which is presumed to be formed via enzymatic
reduction of keto-lactone derivatives of paeoniflorin [19].
The isolated 16 monoterpene derivatives were further examined for
their anti-allergic activity by testing inhibitory effects against DNP-BSA
stimulated β-hexosaminidase release in IgE-sensitized RBL-2H3 cells,
and their IC50 values are shown in Table 2. Cell viability under stimula-
tion was evaluated by the CCK-8 assay and none of the test compounds
at a dose level up to 100.00 μM, nor the positive control Baicalein at a
dose level up to 200.00 μM exhibited observable cytotoxcity. Among
the 16 isolated monoterpenoids, nine compounds showed anti-allergic
activities with IC50 ranging from 41.17 to 87.11 μM. The compound 1
exhibited the most effectiveness with IC50 of 41.17 μM, followed by
compounds 8 (IC50: 42.22 μM) and 11 (IC50: 50.05 μM). Whereas,
compounds 2–5, 7, 10 and 15 showed no inhibitory activity. The results
showed that the basic skeletons of compound 1 and the paeoniflorin-
type (6–14) were important for the activity, compared to the
albiflorin-type (3–5) with a lactone ring in the pinane aglycone. The
similar tendency that the paeoniflorin-type skeleton is essential for
the ameliorating effect on scopolamine-induced amnesia rather than
the albiflorin-type has been observed previously [33]. Of the com-
pounds with paeoniflorin-type skeleton, hydroxyl group at C-4 of the
aglycone was required for the activity, since methylation at this position
resulted in complete losing of activity (6 vs. 7). Song et al. also reported
that the methoxy group at C-4 was deleterious for the anticomplement
activity of the paeoniflorin type monoterpene glycosides [34].
Moreover, compound 8 with O-hydroxybenzoyl moiety at C-8 was
more effective than those with p-hydroxybenzoyl moiety (12–14). A
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.fitote.2015.11.011.
References
[1] Jiangsu New Medical College, Glossary of Chinese Herbal Medicines, Shanghai
Science and Technology Press, Shanghai, 1999 1093–1095.
[2] T. Namba, 2nd ed., The Encyclopedia of Wakan-Yaku (Traditional Sino-Japanese
Medicines) with Color Pictures, Vol.I, Hokusha Publishing Co. Ltd., Osaka, 1993
102–104.
[3] The State Pharmacopeia Commission of China, Pharmacopeia of the People's Repub-
lic of China, vol 1 (English version), Medical Science Press, Beijing, China, 2010
306–308.
[4] S. Zhu, X.L. Yu, Y.Q. Wu, F. Shiraishi, N. Kawahara, K. Komatsu, Genetic and chemical
characterization of white and red peony root derived from Paeonia lactiflora, J. Nat.
Med. 69 (2015) 35–45.
[5] J.W. Duan, J.Y. Yang, L.X. Chen, L.J. Zhang, Z.H. Jiang, X.D. Cai, X. Zhang, F. Qiu, Mono-
terpenes from Paeonia albiflora and their inhibitory activity on nitric oxide produc-
tion by lipopolysaccharide-actived microglia, J. Nat. Prod. 72 (2009) 1579–1584.
[6] Y.C. Juan, C.C. Chang, W.J. Tsai, Y.L. Lin, Y.S. Hsu, H.K. Liu, Pharmacological evaluation
of insulin mimetic novel suppressors of PEPCK gene transcription from Paeoniae
Rubra Radix, J. Ethnopharmacol. 137 (2011) 592–600.
[7] M.J. Chuang, J.K. Sohng, D.J. Choi, Y.I. Park, Inhibitory effect of phloretin and
biochanin
A on IgE-mediated allergic responses in rat basophilic leukemia
RBL-2H3 cells, Life Sci. 93 (2013) 401–408.
[8] C.N. He, Y. Peng, Y.C. Zhang, L.J. Xu, J. Gu, P.G. Xiao, Photochemical and biological
studies of Paeoniaceae, Chem. Biodivers. 7 (2010) 805–838.
[9] B. Lee, Y.W. Shin, E.A. Bae, S.J. Han, J.S. Kim, S.S. Kang, D.H. Kim, Antiallergic effects of
the root of Paeoniae lactiflora and its constituents paeoniflorin and paeonol, Arch.
Pharm. Res. 31 (2008) 445–450.
[10] N. Kageyama-Yahara, Y. Suehiro, F. Maeda, S. Kageyama, J. Fukuoka, T. Katagiri, T.
Yamamoto, M. Kadowaki, Pentagalloylglucose down-regulates mast cell surface
FcεRI expression in vitro and in vivo, FEBS Lett. 584 (2010) 111–118.
[11] S.H. Kim, C.D. Jun, K. Suk, B.J. Choi, S. Park, S.H. Lee, H.Y. Shin, D.K. Kim, T.Y. Shin,
Gallic acid inhibits histamine release and pro-inflammatory cytokine production
in mast cell, Toxicol. Sci. 91 (2006) 123–131.
[12] I.M. Roitt, J. Brosttoff, D.K. Male, Immunology, Fifth edition Mosby, London, 1998,
ISBN 0 7234 2918 9.
[13] T. Kawakami, S.J. Galli, Regulation of mast-cell and basophil function and survival by
IgE, Nat. Rev. Immunol. 2 (2002) 773–786.
[14] A. Sato, H. Tamura, High antiallergic activity of 5, 6, 4′-trihydroxy-7, 8, 3′-
trimethoxyflavone and 5, 6-dihydroxy-7, 8, 3′, 4′-tetramethoxyflavone from eau
de cologne mint (Mentha×pipertia citrata), Fitoterapia 102 (2015) 74–83.
[15] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, I. Kouno, Facile discrimination of aldose
enantiomers by reversed-phase HPLC, Chem. Pharm. Bull. 55 (2007) 899–901.
[16] S.S. Hong, W. Jeong, J.K. Kim, J.G. Kwon, J.Y. Lee, E.K. Ahn, J. Oh, D.W. Seo, J.S. Oh,
Neolignan inhibitors of antigen-induced degranulation in RBL-2H3 cells from the
needles of Pinus thunbergii, Fitoterapia 99 (2014) 347–351.
[17] Traditional Medical & Pharmaceutical Database: http://dentomed.u-toyama.ac.jp/en/
[18] H. Matsuda, T. Morikawa, K. Ueda, H. Managi, M. Yoshikawa, Structural require-
ments of flavonoids for inhibition of antigen-induced degranulation, TNF-α and
IL-4 production from RBL-2H3 cells, Bioorg. Med. Chem. 10 (2002) 3123–3128.
[19] M. Kaneda, Y. Iitaka, S. Shibata, Chemical studies on the oriental plant drugs-XXXIII: the
absolute structures of paeoniflorin, albiflorin, oxypaeoniflorin and benzoylpaeoniflorin
isolated from Chinese paeony root, Tetrahedron 28 (1972) 4309–4317.
[20] Y.B. Ma, D.G. Wu, J.K. Liu, Paeonivayin, A new monoterpene glycoside from Paeonia
delavayi, Chin. Chem. Lett. 10 (1999) 771–774.
Table 2
Inhibitory effects of 16 Monoterpene derivatives against DNP-BSA stimulated β-hexosamin-
idase release in IgE-sensitized RBL-2H3 cells.
Compound no.
IC50 (μM)
Compound no.
IC50 (μM)
[21] A. Braca, P.V. Kiem, P.H. Yen, N.X. Nhiem, T.H. Quang, N.X. Cuong, C.V. Minh, New
monoterpene glycosides from Paeonia lactiflora, Fitoterapia 79 (2008) 117–120.
[22] M. Okasaka, Y. Kashiwada, O. Kodzhimatov, O. Ashrmetov, Y. Takaishi, Monoterpene
glycosides from Paeonia hybrida, Phytochemistry 69 (2008) 1767–1772.
[23] L.Q. Ding, Z.H. Jian, Y. Liu, L.X. Chen, Q. Zhao, X.S. Yao, F. Zhao, F. Qiu, Monoterpene
inhibitors of NO production from Paeonia suffruticosa, Fitoterapia 83 (2012)
1598–1603.
1
6
8
9
11
12
41.17 3.2c
76.20 5.5
42.22 1.8
75.44 2.5
55.05 3.9
87.11 1.1
13
14
16
77.51 2.0
76.68 4.6
59.88 3.7
NAa
2–5, 7, 10, 15
Baicaleinb
37.78 1.0
[24] S.S. Kang, K.H. Shin, H.J. Chi, Galloylpaeoniflorin, a new acylated monoterpene gluco-
a
IC50 values are more than 100 μM.
side from paeony root, Arch. Pharm. Res. 14 (1991) 52–54.
b
c
Positive control [18].
[25] R.B. An, H.C. Kim, S.H. Lee, G.S. Jeong, D.H. Sohn, H. Park, D.Y. Kwon, J.H. Lee, Y.C. Kim,
A new monoterpene glycoside and antibacterial monoterpene glycosides from
Paeonia suffruticosa, Arch. Pharm. Res. 29 (2006) 815–820.
The results are expressed as the mean S.D., IC50 values were calculated by Probit
regression analysis of SPSS software (version 15.0).

Y.-H. Shi et al. / Fitoterapia 108 (2016) 55–61
61
[26] N. Murakami, M. Saka, H. Shimada, H. Matsuda, J. Yamahara, M. Yoshikawa, New
bioactive monoterpene glycosides from Paeoniae Radix, Chem. Pharm. Bull. 44
(1996) 1279–1281.
[27] M. Yoshikawa, T. Ohta, A. Kawaguchi, H. Matsuda, Bioactive constituents of Chinese
natural medicines. V. Radical scavenging effect of moutan cortex (1): absolute
stereostructures of two monoterpenes, paeonisuffrone and paeonisuffral, Chem.
Pharm. Bull. 48 (2000) 1327–1331.
[28] M. Shimizu, T. Hayashi, N. Morita, F. Kiuchi, H. Noguchi, Y. Iitaa, U. Sankawa, The
structure of paeoniflorigenone, a new monoterpene isolated from Paeoniae Radix,
Chem. Pharm. Bull. 31 (1983) 577–583.
[29] P.H. Yen, P.V. Kiem, N.X. Nhiem, N.H. Tung, T.H. Quang, C.V. Minh, J.W. Kim, E.M.
Choi, Y.H. Kim, A new monoterpene glycoside from the roots of Paeonia lactiflora in-
crease the differentiation of osteoblastic MC3T3-E1, Arch. Pharm. Res. 30 (2007)
1179–1185.
[30] M.H. Yean, J.Y. Lee, J.S. Kim, S.S. Kang, Phytochemical studies on paeoniae radix (1)-
monoterpene glucosides, Kor. J. Pharmacogn. 39 (2008) 19–27.
[31] H.J. Chung, R.J. Ferl, Arabidopsis alcohol dehydrogenase expression in both shoots
and roots is conditioned by root growth environment, Plant Physiol. 121 (1999)
429–436.
[32] C.E. Thompson, C.L. Fernandes, O.N. de Souza, L.B. de Freitas, F.M. Salzano, Evaluation
of the impact of functional diversification on Poaceae, Brassicaceae, Fabaceae, and
Pinaceae alcohol dehydrogenase enzymes, J. Mol. Model. 16 (2010) 919–928.
[33] A.A. Abdel-Hefez, M.R. Meselhy, N. Nakamura, M. Hattori, H. Watanabe, Y.
Murakami, M.A. El-gendy, N.M. Mahfouz, T.A. Mohamed, Effects of paeoniflorin de-
rivatives on scopolamine-induced amnesia using a passive avoidance task in mice;
structure-activity relationship, Biol. Pharm. Bull. 21 (1998) 1174–1179.
[34] W.H. Song, Z.H. Cheng, D.F. Chen, Anticomplement monoterpenoid glucosides from
the root bark of Paeonia suffruticosa, J. Nat. Prod. 77 (2014) 42–48.