Monoterpene glycosides with anti-inflammatory activity from Paeoniae Radix

Article abstract of DOI:10.1016/j.fitote.2019.104290

Six new monoterpene glycosides, named 6′-O-nicotinoylalbiflorin (1), 4′-O-vanillylalbiflorin (2), paeonidanin L (3), paeoniflorigenin-1-O-β-D-xyloside (4), 6′-(2-hydroxypropanoyl)-paeoniflorin (5), oxylactiflorin (6), together with 16known ones (7–22) were isolated from the 70% ethanol extract of Paeoniae Radix. Their structures were elucidated based on spectroscopic analysis (1D and 2D NMR, HRESIMS, IR and UV), chemical evidences and comparison with literatures. The inhibitory effects of all the isolates were evaluated against lipopolysaccharide (LPS) stimulated PGE₂ production in RAW 264.7 macrophages.

Full text of DOI:10.1016/j.fitote.2019.104290

Fitoterapia138(2019)104290
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Monoterpene glycosides with anti-inflammatory activity from Paeoniae
Radix
Hai-bo Lia,1, Ying Shia,1, Qianqian Panga, Yudan Meia, Zhenzhen Sub, Xin-Sheng Yaoa,⁎
,
Yang Yua,⁎
a Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM
and New Drug Research, Jinan University, Guangzhou 510632, China
b Kanion Pharmaceutical Co. Ltd., State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Six new monoterpene glycosides, named 6′-O-nicotinoylalbiflorin (1), 4′-O-vanillylalbiflorin (2), paeonidanin L
(3), paeoniflorigenin-1-O-β-D-xyloside (4), 6′-(2-hydroxypropanoyl)-paeoniflorin (5), oxylactiflorin (6), together
with 16known ones (7–22) were isolated from the 70% ethanol extract of Paeoniae Radix. Their structures were
elucidated based on spectroscopic analysis (1D and 2D NMR, HRESIMS, IR and UV), chemical evidences and
comparison with literatures. The inhibitory effects of all the isolates were evaluated against lipopolysaccharide
(LPS) stimulated PGE2 production in RAW 264.7 macrophages.
Paeoniae Radix
Monoterpene glycosides
Anti-inflammatory
PGE2
1. Introduction
(2), paeonidanin L (3), paeoniflorigenin-1-O-β-D-xyloside (4), 6′-(2-hy-
droxypropanoyl)-paeoniflorin (5), oxylactiflorin (6), together with 16
The peony is a perennial herb in Paeonia, the single genus of the
family Paeoniaceae. Peony roots (“Shaoyao” in Chinese) are dis-
tinguished by their red or white color into two therapeutic herbal
products, Radix Paeoniae Rubra (Chishao) and Radix Paeoniae Alba
(Baishao). Chishao, red peony root, is brownish in appearance and of-
ficially originated from either P. lactiflora Pall. or P. veitchii Lynch with
traditional usage for eliminating stasis and invigorating blood circula-
tion. While baishao is white and peeled in appearance, only P. lactiflora
Pall is officially considered, with claims of its nourishing blood, sub-
duing hyperactivity and regulating menstruation [1–4]. There have
been many researches on phytochemical constituents of peony roots,
including monoterpenoids and their glycosides, flavonoids, hydro-
lysable tannins (polyphenols), and triterpenoids [5]. The “cage-like”
monoterpene derivatives (paeoniflorin analogues) are considered as the
main and characteristic bioactive components. These compounds have
greater potentials for neuroprotective, anti-proliferative, anti-allergic,
anti-coagulant, anti-inflammatory and anti-diabetic activities, etc.
[6–13].
known analogues which were identified as albiflorin (7) [14], paeoni-
vayin (8) [15], 4-O-galloylalbiflorin (9) [16], paeoniflorin (10) [14],
oxypaeoniflorin (11) [14], mudanpioside E (12) [17], benzoylpaeoni-
florin (13) [14], 6’-O-vanillylpaeoniflorin (14) [18], galloylpaeoniflorin
(15) [19], 4′-O-galloylpaeoniflorin (16) [20], 4′-O-benzoylpaeoniflorin
(17) [16], 4-O-galloylpaeoniflorin (18) [21], isomaltopaeoniflorin (19)
[18], mudanpioside C (20) [17], lactiflorin (21) [22], 1-O-β-D-gluco-
pyranosyl-8-O-benzoylpaeonisuffrone (22) [9].
Herein, we report the isolation and structure elucidation, and anti-
inflammatory activity of these compounds.
2. Experimental
2.1. General experimental procedures
1H and 13C NMR spectra were measured on a Bruker AV 600 (Bruker
Co. Ltd., Bremen, German) at 600 and 150 MHz with solvent signals
(CD3OD, δH 3.30/δC 49.0) as internal reference. IR spectra were taken
on a JASCO FT/IRBCA plus spectrometer (JASCO International Co. Ltd.,
Tokyo, Japan) with KBr. UV spectra were acquired using a JASCO V-
550 UV/Vis spectrometer (JASCO International Co. Ltd., Hachioji,
Tokyo, Japan). Optical rotation was measured on JASCO P-1020 digital
In our continuing efforts to seek the bioactive monoterpenes from
genus Paeonia, a re-investigation on Radix Paeoniae Alba led to the
discovery of twenty-two monoterpene derivatives (Fig. 1), including
6new ones, namely 6′-O-nicotinoylalbiflorin (1), 4′-O-vanillylalbiflorin
⁎ Corresponding authors at: College of Pharmacy, Jinan University, 601 Huangpu Avenue, Guangzhou 510632, PR China.
E-mail addresses: tyaoxs@jnu.edu.cn (X.-S. Yao), 1018yuyang@jnu.edu.cn (Y. Yu).
1 Both authors contributed equally to this work.
https://doi.org/10.1016/j.fitote.2019.104290
Received 16 July 2019; Received in revised form 5 August 2019; Accepted 5 August 2019
Availableonline06August2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

H.-b. Li, et al.
Fitoterapia138(2019)104290
Fig. 1. Chemical Structures of Compounds 1–22 (red: new). (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
polarimeter (JASCO International Co. Ltd., Hachioji, Tokyo, Japan).
HR-ESI-MS spectra were measured on a Waters Synapt G2 mass spec-
trometer (Waters, Manchester, U. K.) with a RP-18 column (1.7 μm, ϕ
3.0 × 150 mm; BEH). HPLC analyses were performed on a Waters 2695
separations module (Waters, Manchester, U.K.) equipped with a 2998
photodiode array (PDA) detector and an Alltech 3300 evaporative light
scattering detector (ELSD; Alltech Inc., Deerfield, Illinois, U.S.A.) using
a Phenomenex Gemini C18 column (5 μm, ϕ 4.6 × 250 mm; FLM Inc.,
Guangzhou, China). The semi-preparative HPLC analyses were per-
formed on a Waters 1515 isocratic HPLC pump (Waters, Manchester,
U.K.) coupled with a 2489 UV/Vis detector (Waters, Manchester, U.K.)
and a Phenomenex Gemini C18 column (5 μm, ϕ 10 × 250 mm; FLM
Inc., Guangzhou, China).
The methanol of HPLC grade was purchased from BCR International
Co. Ltd. (Shanghai, China), while the acetonitrile was purchased from
Merck (Darmstadt, Germany). Diaion HP-20 (Mitsubishi Chemical Co.,
Tokyo, Japan), silica gel (200–300 mesh, Qingdao Marine Chemical
Ltd., Shandong, China), ODS-silica gel (12 nm, S-50 μm, YMC Ltd.,
Tokyo, Japan) and Sephadex LH-20 (Amersham Pharmacia Biotech,
Sweden) were used for chromatography column (CC). TLC was per-
formed on pre-coated silica gel plate (SGF254, 0.2 mm, Yantai Chemical
Industry Research Institute, Shandong, China).
tR = 32.2 min, 22% CH3CN (0.1% formic acid)] and 12 [30.0 mg,
tR = 29.0 min, 22% CH3CN (0.1% formic acid)], respectively. Fr. 2D10
(9.7 g) was purified over ODS (CH3OH-H2O, 40% to 100%, v/v) to af-
ford compounds 10 [1.6 g, 60% CH3OH] and 19 [200.3 mg, 50%
CH3OH].
Fr.2E (8.5 g) was subjected to an ODS CC (CH3OH-H2O, 25% to
100%, v/v) to afford 14 fractions (Fr 2E1 to 2E14). Fr. 2E4 (1.6 g) was
separated over a silica gel CC (EtOAc-CH3OH, 9:1, v/v), followed by an
ODS CC (CH3OH-H2O, 30%)，and further purified by a preparative
HPLC to yield compounds 4 [15.9 mg, tR = 12.1 min, 25% CH3OH
(0.1% formic acid)], 6 [25.3 mg, tR = 16.7 min, 25% CH3OH (0.1%
formic acid)], 5 [30.9 mg, tR = 20.7 min, 25% CH3OH (0.1% formic
acid)], 21 [102.1 mg, tR = 25.2 min, 25% CH3CN (0.1% formic acid)]
and 1 [15.6 mg, tR = 30.6 min, 25% CH3CN (0.1% formic acid)]. Fr 2E7
(543.5 mg) and 2E10 (27.6 mg) were chromatographed by Sephadex
LH-20 CC (CH3OH-H2O, 7:3, v/v), and then subjected to preparative
HPLC to yield compounds 11 [12.0 mg, tR = 27.9 min, 20% CH3CN
(0.1% formic acid)] and 3 [32.2 mg, tR = 48.2 min, 22% CH3CN (0.1%
formic acid)], respectively. Compounds 2 [22.6 mg, tR = 32.3 min, 22%
CH3CN (0.1% formic acid)] and 15 [16.5 mg, tR = 27.9 min, 22%
CH3CN (0.1% formic acid)] were obtained from Fr 2E8 (192.1 mg) by
ODS CC (CH3OH-H2O, 40% to 100%, v/v) followed by a preparative
HPLC.
Fr·2H (161.0 g) was chromatographed by Sephadex LH-20 CC
(CH3OH-H2O, 1:1, v/v) to give 8 subfractions (Fr. 2H1-2H8). Fr. 2H5
(53.2 g) precipitated crystal compound 18 (30.2 g). Fr. 2H6 (2.3 g) was
chromatographed by ODS CC using 45% CH3OH to afford 8 fractions
(Fr 2H6A-2H6H). Fr.2H6A (0.8 g), 2H6C (43.9 mg) and 2H6F (46.2 mg)
were further purified by preparative HPLC to obtain compounds 13
[200 mg, tR = 27.2 min, 25% CH3OH (0.1% formic acid)], 14 [2.1 mg,
tR = 36.7 min, 25% CH3OH (0.1% formic acid)], 20 [37.6 mg,
tR = 39.6 min, 25% CH3OH (0.1% formic acid)] and 16 [201.3 mg,
tR = 32.5 min, 25% CH3OH (0.1% formic acid)]. Fr·2H8 (11.4 g) was
chromatographed by MCI CC using a gradient elution (CH3OH-H2O,
from 15% to 65%, v/v) to afford 7 fractions (Fr 2H8A to 2H8G). Fr.
2H8B (492.0 mg) was further purified by preparative HPLC to obtain
compounds 8 [30.2 mg, tR = 22.1 min, 25% CH3OH (0.1% formic
acid)] and 9 [62.1 mg, tR = 28.0 min, 25% CH3OH (0.1% formic acid)].
Fr. 2 K (2.3 g) was chromatographed by ODS CC using a 45% CH3OH
system to afford compound 17 (200.2 mg).
2.2. Plant materials
Paeoniae Radix Alba were collected in Anhui, China, and purchased
from Hubei tianji traditional Chinese medicine decoction piece Co. LTD.
in Nov, 2016, and identified by Prof. G. X. Zhou of Jinan University. A
sample (No. 20160601) is deposited in Jinan University, Guangzhou,
China.
2.3. Extraction and isolation
Paeoniae Radix Alba (29.0 kg) were extracted with 70% EtOH-H2O
(120 L, v/v) by heat reflux for 2 times (2 h each) to afford a total extract
(PRA, 4.0 kg), which was suspended in water and separated by HP-20
resin column chromatography (CC) eluted with EtOH-H2O (15:85,
50:50, 95:5) gradient to afford 3 fractions (PRA-1 to PRA-3). Fr. PRA-2
(600.0 g) was separated over a silica gel column eluting with a CH2Cl2-
CH3OH gradient (from 95:5 to 0:100, v/v) to afford 14 fractions (Fr. 2A
to 2 N). Fr. 2D (20.3 g) was chromatographed by ODS CC (CH3OH-H2O,
from 25% to 100%, v/v) to afford 12 fractions (Fr. 2D1 to 2D12). Fr.
2D5, 8, 9 were subjected to preparative HPLC to yield compounds 7
[10.3 mg, tR = 15.2 min, 15% CH3CN (0.1% formic acid)], 22 [30.0 mg,
2.4. Data for structure elucidation of new compounds
28
6′-O-nicotinoylalbiflorin (1): White amorphous powder；[α]D
-
2

H.-b. Li, et al.
Fitoterapia138(2019)104290
3

H.-b. Li, et al.
Fitoterapia138(2019)104290
100.7 (c 0.5, CH3OH); UV (CH3OH) λmax (logε): 202 (3.74), 227 (3.93),
264 (3.08), 271 (3.01) nm; IR (KBr) νmax: 3443, 2920, 2555, 1772,
1281, 1067, 719 cm−1; HR-ESI-MS: m/z 586.1929 [M + H]+ (calcd.
For C29H31N1O12 586.1925); 1H and 13C NMR data see Table 1.
4′-O-vanillylalbiflorin (2): White amorphous powder; [α]D28-34.9 (c
0.5, CH3OH); UV (CH3OH) λmax (logε): 204 (3.99), 224 (3.94), 266
(3.54), 297 (3.32) nm; IR (KBr) νmax: 3443, 2920, 1760, 1612, 1346,
710 cm−1; HR-ESI-MS: m/z 631.2026 [M + H]+ (calcd. For C31H34O14
631.2027); 1H and 13C NMR data see Table 1.
2.6. Anti-inflammatory activity assay
Cell culture and Viability: RAW 264.7 murine macrophage cell line
was obtained from Chinese Academy of Sciences. The cells were grown
in DMEM (Gibico, USA) containing 10% FBS (Gibico, USA), 100 U/mL
penicillin and 100 μg/mL streptomycin. They were cultured at 37 °C in
5% CO2. Cell viability was analyzed using MTT assay. Compounds were
added to the cells and incubated for 1 h and then cells were treated with
or without LPS for 18 h at 37 °C with 5% CO2. MTT solution (5 g/L) was
added to each well and incubated for 4 h at 37 °C. The formazan dyes in
the cells were dissolved in 100 μL 10% SDS-HCl solution. The optical
density was read at 570 nm (reference, 650 nm) using a microplate UV/
vis spectrophotometer (Tecan, Mannedorf, Switzerland). The cell via-
bility in the control group (cells were not treated by compounds and
LPS) was set as 100%.
Effect of compounds 1–22 on the PGE2 production: Cells were
seeded in a 96-well plate at a density of 1 × 104 cells/well in DMEM
and incubated for 24 h. The cells were pretreated with compounds for
30 min and treated with LPS (1 μg/mL) for 24 h in the presence or ab-
sence of different concentrations of the compounds. The concentration
of PGE2 in the culture medium was determined using commercial ELISA
kits according to the instructions. The IC50 values were calculated from
calibration curves of the absorbance inhibition by each compound at
three concentrations against the increasing absorbance of macrophage
cells stimulated only with LPS (without the compounds). Data were
obtained from three independent experiments (n = 3).
paeonidanin L (3): White amorphous powder; [α]D25-87.1 (c 0.35,
CH3OH); UV (CH3OH) λmax (logε): 203 (4.46), 224 (4.64), 281 (4.11)
nm; IR (KBr) νmax: 3440, 2923, 1705, 1606, 1343, 716 cm−1; HR-ESI-
MS: m/z 759.1829 [M + Na]+ (calcd. For C37H36O16 759.1901); 1H
and 13C NMR data see Table 1.
paeoniflorigenin-1-O-β-D-xyloside (4): White amorphous powder;
[α]D28-32.0 (c 0.5, CH3OH); UV (CH3OH) λmax (logε): 202 (3.82), 230
(4.01), 274 (2.89); IR (KBr) νmax: 3426, 2978, 2929, 1711, 1444, 1269,
1073, 716 cm−1; HR-ESI-MS: m/z 473.1422 [M + Na]+ (calcd. For
C
22H26O10 473.1424); 1H and 13C NMR data see Table 1.
6′-(2-hydroxypropanoyl)-paeoniflorin (5): White amorphous
powder; [α]D28-31.7 (c 0.5, CH3OH); UV (CH3OH) λmax (logε): 203
(4.14), 230 (4.36), 274 (3.22), 282 (3.12); IR (KBr) νmax: 3426, 2929,
1711, 1444, 1269, 1073, 716 cm−1
; HR-ESI-MS: m/z 575.1743
[M + Na]+ (calcd. For C26H32O13 575.1741); 1H and 13C NMR data see
Table 1.
oxylactiflorin (6): White amorphous powder; [α]D25-96.9 (c 0.5,
CH3OH), UV (CH3OH) λmax (logε): 203 (3.97), 210 (3.86), 260 (4.00)，
IR (KBr) νmax: 3538, 3294, 2932, 2891, 1769, 1453, 1267, 1125, 1021,
713 cm−1。HR-ESI-MS m/z 501.1368 [M + H]+ (calcd. for C23H26O11
501.1373)，1H and 13C NMR data see Table 1.
3. Results and discussion
3.1. Structure elucidation of new compounds
Compound 1 was isolated as a white amorphous powder. The HR-
ESI-MS of 1 displayed a quasi-molecular ion peak at m/z 586.1929
[M + H]+ (calcd. For 586.1925), corresponding to the molecular for-
mula C29H31N1O12. The analyses of NMR spectra revealed that 1 pos-
sessed 29 carbons. Among them, 23 carbon signals were assigned to a
2.5. Acidic hydrolysis and sugar analysis
The absolute configurations of the sugar moieties in the structures
were determined by the method of Tanaka et al. [23]. Each compound
(1.0–2.0 mg) was refluxed with 2 mL of 2 M HCl for 2 h at 90 °C. The
hydrolysates were extracted with equal volume of ethyl acetate twice.
The aqueous layer was dried, and then reacted with 2.5 mg L-cysteine
methyl ester hydrochloride in 1 mL of pyridine for 1 h at 60 °C. A total
of 5 μL o-tolylisothiocyanate was added to the above mixture for 1 h at
60 °C. The reaction products were filtered by a 0.45 μm filter membrane
for HPLC analysis at 35 °C with isocratic elution of 25% CH3CN con-
taining 0.1% formic acid for 30 min. Phenomenex Gemini C18 column
(5 μm, ϕ 4.6 × 250 mm; FLM Inc., Guangzhou, China) was used to
analysis at 0.8 mL/min flow rate. Peaks were detected by UV detector at
250 nm. The peaks of authentic samples of D-glucopyranose, L-gluco-
pyranose, D-xylose and L-xylose after treatment in the same manner
were detected at 19.8, 18.0, 23.0 and 21.2 min, respectively.
C
10-monoterpene aglycone [δC 86.9 (C-1), 93.2 (C-2), 41.6 (C-3), 68.2
(C-4), 41.6 (C-5), 56.6 (C-6), 28.3 (C-7), 61.9 (C-8), 177.8 (C-9), 20.5
(C-10)], a hexosyl [δC 100.0 (C-1′), 74.8 (C-2′), 77.8 (C-3′), 71.7 (C-4′),
75.3 (C-5′), 65.4 (C-6′)], and a benzoyl unit [δC 131.2 (C-1′′), 130.7 (C-
2′′, 6′′), 129.6 (C-3′′, 5′′), 134.4 (C-4′′) and 167.9 (C-7′′)], assembling
into a main structure fragment as albiflorin (compound 7). The re-
maining 6 carbon signals, including 5 sp2 phenyl carbons and one
carbonyl, in combination with unsaturation degrees and a nitrogen
atom, suggested the presence of a nicotinoyl moiety in 1. This deduc-
tion was further supported by carbon chemical shifts, 1H-1H COSY and
HMBC spectral data. The 1H-1H COSY spectrum disclosed interactions
of H-4′′′/H-5′′′/H-6′′′, associated with a series of HMBC correlations of
H-5′′′/C-3′′′, H-4′′′/C-2′′′, C-7′′′ and H-2′′′/C-6′′′, C-4′′′, C-7′′′, un-
doubtedly allowed the assignment of a nicotinoyl group. Additionally,
the nicotinoyl moiety (carbonyl carbon at δC 165.9) was determined to
Fig. 2. Key HMBC and ROESY correlations of 1, 3 and 5.
4

H.-b. Li, et al.
Fitoterapia138(2019)104290
Fig. 3. Inhibitory effect of compounds 1, 2, 10, 13, 17 and 18 on PGE2 production induced by LPS in macrophages (IC50 Values Expressed in μM).
connect to C-6 of glucosyl due to the obvious downfield shift of C-6 (δC
65.4) and HMBC correlations of H-6′/C-7′′′. The configuration of the
monoterpene aglycone in 1 was considered as the same as the well-
known paeoniflorin-type monoterpene glucosides in ROESY experiment
(H3-10/H2-3, H-3α/H-7α, H-4/H-7α, H-7α/H-1′ and H-7β/H-8b (see
Fig. 2). Thus, the structure of 1 was elucidated as 6′-O-nicotinoylalbi-
florin.
Compound 2 was obtained as a white amorphous powder with a
molecular formula of C31H34O14 based on HR-ESI-MS (m/z 631.2026
[M + H]+, calcd. For 631.2027). Detailed inspection of the NMR
spectroscopic data of 2 indicated it has the similar structure as com-
pound 9 (4-O-galloylalbiflorin) [14]. The difference was found in the
presence of the vanilly signals [δH 6.82 (1H, d, J = 8.0 Hz, H-5′′′), 7.53
(1H, d, J = 1.8 Hz, H-5′′′), 7.55 (1H, dd, J = 8.0, 1.8 Hz, H-6′′′), δC
122.1 (C-1′′′), 114.0 (C-2′′′), 148.7 (C-3′′′), 153.0 (C-4′′′), 115.9 (C-5′′′),
125.5 (C-6′′′) and 167.1 (C-7′′′)] in NMR spectra of 2. Acid hydrolysis of
2 yielded D-glucose by HPLC analysis with authentic samples as the
reference [23]. The HMBC correlation (see Fig. 2) of H-4 with C-7′′′
indicated that the vanilly moiety was connected to C-4. On the basis of
these data, the structure of 2 was settled down and named as 4′-O-
vanillyalbiflorin.
were recognized as a benzoyl group, a monoterpene aglycone and a
glycosidic moiety. Comparison of these NMR data with those of com-
pound 10 (paeoniflorin) suggested that they had only different sugar
unit, which was further confirmed by NMR and acid hydrolysis ex-
periments. Accordingly, the 13C NMR spectroscopic data of the sugar in
4 matched well with a xylose [25]. Acid hydrolysis of 4 [23] followed
by HPLC analysis afforded a D-xylopyranosyl. A β-configuration at C-1′′
in 4 was identified due to a large coupling constant (J = 7.8 Hz) of
anomeric proton. Thus, compound
4 was elucidated as paeoni-
florigenin-1-O-β-D-xyloside.
Compound 5 was obtained as a white amorphous powder, and its
molecular formula of C26H32O13 was elucidated from HR-ESI-MS data
(m/z 575.1743 [M + Na]+, calcd. For 575.1741). Detailed inspection
of the NMR spectroscopic data of 5 indicated it was a derivative of
compound 10 (paeoniflorin) [14]. The difference was found in the
presence of the 2-hydroxypropanoyl signals at [δH 4.27 (2H, dd,
J = 7.0, 2.3 Hz, H-2′′′), 1.38 (3H, d, J = 6.6 Hz, H-3′′′), δC 67.8 (C-2′′′),
20.3 (C-3′′′) and 176.0 (C-1′′′)] in the NMR spectra of 5. The HMBC
correlation from H2–6′/C-1′′′ indicated that the 2-hydroxypropanoyl
moiety was connected at C-6′ (see Fig. 2). Thus, the structure of 5 was
assigned as 6′-(2-hydroxypropanoyl)-paeoniflorin.
Compound 3 was obtained as a white amorphous powder, and its
molecular formula was determined as C37H36O16 according to the HR-
Compound 6, a white amorphous powder, showed the molecular
formula
C23H26O11, as determined by HR-ESI-MS m/z 501.1368
ESI-MS peak at m/z 759.1829 [M + Na]+ (calcd. For C37H36O16
,
[M + Na]+ (calcd. For C23H26O11Na, 501.1373) and NMR data.
Comparing the 1H and 13C NMR spectroscopic data of 6 with those of a
previously reported compound 21 (lactiflorin) [22] revealed the simi-
larity of their chemical shifts, except for the presence of a p-hydro-
xybenzoyl moiety in the structure of 6 instead of the benzoyl moiety as
in lactiflorin. Thus, compound 6 was determined as oxylactiflorin.
759.1901) with twenty degrees of unsaturation. The 1H NMR spectrum
of 3 displayed a methyl group at δH1.38 (3H, s), an acetal proton at δH
6.54 (1H, s), and an anomeric proton at δH 4.69 (1H, d, J = 7.7 Hz). The
13C NMR spectrum displayed 37 carbons including two benzoate moi-
eties [δC 131.2 (C-1′′), 130.5 (C-2′′, 6′′), 129.7 (C-3′′, 5′′), 134.4 (C-4′′),
167.6 (C-7′′) and 131.2 (C-1′′′), 130.5 (C-2′′′, 6′′′), 129.3 (C-3′′′, 5′′′),
134.3 (C-4′′′), 167.5 (C-7′′′)], a 1,2,4,5-tetrasubstituted benzene ring
[δC 120.4 (C-1′′′′), 110.6 (C-2′′′′, 6′′′′), 146.5 (C-3′′′′, 5′′′′), 140.4 (C-4′′′′)
and 167.0 (C-7′′′′)], a set of glycosyl signals at δC 99.9 (C-1′), 75.0 (C-
2′), 77.8 (C-3′), 72.0 (C-4′), 75.4 (C-5′) and 65.2 (C-6′), an acetal carbon
at δC 100.7, and a carbonyl carbon at δC 209.6. The 13C NMR spectrum
was similar to that of paeonidanin A [24], except for the presence of a
galloyl moiety instead of the methoxy group linked to the C-9. The
HMBC correlations from H-3/C-1, C-4, C-5, H-5/C-1, C-4, C-8, C-9, H-7
to C-2, C-4, C-6, H-9/C-2, C-4, C-6, and H-10/C-1, C-2, C-3 were ob-
served. In addition, HMBC correlations from H-1′ to C-1, H-8 to C-7″, H-
6′ to C-7″′ and from H-9 to C-7′′′′ confirmed that the sugar unit was
connected to C-1, the two benzoyl moieties (carbonyl carbons at δC
167.6 and 167.5) were connected to C-8 and C-6′, respectively, and the
galloyl moiety (carbonyl carbon at δC 167.0) was connected to C-9.
Thus, the structure of 3 was elucidated and named as paeonidanin L.
The molecular formula of compound 4, C22H26O10, was established
by HR-ESI-MS (m/z 473.1422 [M + Na]+, calcd. For 473.1424). The
analyses of NMR spectra revealed that 4 possessed 22 carbons, which
3.2. Anti-inflammatory effects of the compounds
All isolated compounds were evaluated for their inhibitory effects
on PGE2 production in macrophages induced by LPS. In addition, cy-
totoxicity in the present experiment was determined in parallel by the
MTT method to find whether inhibition on PGE2 production was due to
the cytotoxicity of the test compounds. Compounds 1–22 exerted
minimal cytotoxicity against Raw 264.7 cells (CC50 > 500 μM). As
shown in Fig. 3, hydrocortisone (IC50 10.5
0.8 μM) was used as
positive control. Compound 18 exhibited strong inhibitory activity on
PGE2 production, while compounds 1, 2, 10, 13 and 17 showed
medium anti-inflammatory activity.
4. Conclusion
In the present research, six new monoterpene glycosides (1–6) and
sixteen known compounds (7–22) were obtained and identified from
Radix Paeoniae Alba. Their inhibitory effects against PGE2 production in
5

H.-b. Li, et al.
Fitoterapia138(2019)104290
anti-inflammatory model were assessed. Although experiments showed
some compounds have biological activities, further investigation of
their therapeutic potential is needed. The diversity for botanical
paeoniflorin compounds identified in this paper have provided a source
for further pharmaceutical drug development.
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Funding resource
[12] J.W. Duan, J.Y. Yang, L.X. Chen, L.J. Zhang, Z.H. Jiang, X.D. Cai, X. Zhang, F. Qiu,
Monoterpenes from Paeonia albiflora and their inhibitory activity on nitric oxide
production by lipopolysaccharide-actived microglia, J. Nat. Prod. 72 (2009)
1579–1584.
[13] 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.
The authors are thankful to National Natural Science Foundation of
China for financial support to this research (No. 81602984 and
81630097).
Declaration of Competing Interest
[14] M. Kaneda, Y. Iitaka, S. Shibata, Chemical studies on the oriental plant drugs-
XXXIII: the absolute structures of paeoniflorin, albiflorin, oxypaeoniflorin and
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Paeonia delavayi, Chin. Chem. Lett. 10 (1999) 771–774.
The authors declare no competing financial interest.
Appendix A. Supplementary data
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