2-Methyl-L-erythritol glycosides from Gardenia jasminoides

Article abstract of DOI:10.1016/j.fitote.2013.05.018

Two new glycosides, 2-methyl-L-erythritol-4-O-(6-O-trans-sinapoyl)-β- D-glucopyranoside (1) and 2-methyl-L-erythritol-1-O-(6-O-trans-sinapoyl)-β- D-glucopyranoside (2), along with two known triterpenoids (3-4), four quinic acid derivatives (5-8) and one flavonoid (9) were isolated from the fruit of Gardenia jasminoides. Their structureswere elucidated throughMS and 2DNMR experiments (HMQC and HMBC). Inhibitory effects of the isolated compounds on nitric oxide production in lipopolysaccharide-activated macrophages were evaluated. Though 2-methyl-D-erythritol and its glycosides have been reported in a few references, this is the first report about 2-methyl-L-erythritol glycosides. Based on this finding,wepropose that 2-methyl-L-erythritolmight be a new intermediate in the non-mevalonate biosynthesis of terpenoids.

Full text of DOI:10.1016/j.fitote.2013.05.018

Fitoterapia 89 (2013) 126–130
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
2
-Methyl-L-erythritol glycosides from Gardenia jasminoides
a,b,1
, Kaifeng Peng a,b,1, Shizhe Zhao a,b, Feng Zhao c,
Liguo Yang
a,b,
a,b,
Lixia Chen ⁎, Feng Qiu ⁎⁎
a
Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
School of Pharmacy, Yantai University, No. 32 Road QingQuan, Laishan District, Yantai 264005, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new glycosides, 2-methyl-L-erythritol-4-O-(6-O-trans-sinapoyl)-β-D-glucopyranoside (1) and
2-methyl-L-erythritol-1-O-(6-O-trans-sinapoyl)-β-D-glucopyranoside (2), along with two known
triterpenoids (3–4), four quinic acid derivatives (5–8) and one flavonoid (9) were isolated from the
fruit of Gardenia jasminoides. Their structures were elucidated through MS and 2D NMR experiments
Received 21 August 2012
Accepted in revised form 22 May 2013
Available online 31 May 2013
(
HMQC and HMBC). Inhibitory effects of the isolated compounds on nitric oxide production in
Keywords:
Gardenia jasminoides
lipopolysaccharide-activated macrophages were evaluated. Though 2-methyl-D-erythritol and its
glycosides have been reported in a few references, this is the first report about 2-methyl-L-erythritol
glycosides. Based on this finding, we propose that 2-methyl-L-erythritol might be a new intermediate
in the non-mevalonate biosynthesis of terpenoids.
2-Methyl-L-erythritol glycosides
Triterpenoid
Quinic acid derivatives
Flavonoid
©
2013 Elsevier B.V. All rights reserved.
Anti-inflammatory activity
1
. Introduction
physiological mechanisms in vivo, and a number of studies
have shown that overproduction of NO by NOS is responsible
for inflammation [10]. As a part of our program to investigate
the anti-inflammatory constituents from this plant, chemical
investigation of 60% EtOH extracts of fruit of G. jasminoides was
undertaken. Though 2-methyl-D-erythritol glycosides have
been found ten years ago [11], no reports have been published
about 2-methyl-L-erythritol glycosides. In this study, two new
The dried ripe fruit of Gardenia jasminoides, an important
traditional Chinese medicine (TCM), has been recorded as
Fructus Gardeniae (Chinese herbal name is “zhi zi”) in Chinese
Pharmacopoeia and widely used for the treatment of diuretic,
cholagogue, anti-inflammatory and antipyretic effects [1]. The
phytochemical constituents of G. jasminoides have been
extensively investigated and led to the isolation of iridoids
and their glycosides [2,3], monocyclic monoterpenoids and
their glycosides [4,5], flavonoids [6], crocetin and its glycosides
2
(
-methyl-L-erythritol glycosides, 2-methyl-L-erythritol-4-O-
6-O-trans-sinapoyl)-β-D-glucopyranoside (1) and 2-methyl-
L-erythritol-1-O-(6-O-trans-sinapoyl)-β-D-glucopyranoside
2), along with two known triterpenoids (3–4), four quinic acid
[
[
7,8], and quinic acid derivatives and vanillic acid glycosides
9]. Nitric oxide (NO) is derived from L-arginine by nitric oxide
(
derivatives (5–8) and one flavonoid (9) were isolated from the
fruit of G. jasminoides. In the present research, we report the
isolation and structural elucidation of the new compounds and
the inhibitory effects of all compounds on NO production in
LPS-activated macrophages.
synthase (NOS) in numerous mammalian cells and tissues. As a
well-known intracellular and intercellular signaling molecule,
it plays an important role in the regulation of diverse
⁎
Correspondence to: L. Chen, Department of Natural Products Chemistry,
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, Shengyang 110016, China. Tel.: +86 24 23986515.
2. Experimental
⁎⁎ Correspondence to: F. Qiu, Department of Natural Products Chemistry, school
of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shengyang 110016, China. Tel.: +86 24 23986463; fax: +86 24 23993994.
E-mail addresses: syzyclx@163.com (L. Chen),
2
.1. General
Optical rotations were measured with a Perkin-Elmer 241
fengqiu20070118@163.com (F. Qiu).
1
Contributed equally to this work.
polarimeter. UV spectra were recorded on a Shimadzu UV 2201
0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2013.05.018

L. Yang et al. / Fitoterapia 89 (2013) 126–130
127
spectrophotometer. IR spectra were conducted on a Bruker IFS
5 spectrometer. NMR experiments were performed on Bruker
1633, 1604, 1516, 1457, 1427, 1383, 1285, 1157, 1114,
−
1
1
13
5
828 cm
;
H and C NMR data: see Table 1; HRESIMS m/z
+
ARX-300 and AV-600 spectrometers. The chemical shifts are
stated relative to TMS and expressed in δ values (ppm), with
coupling constants reported in Hz. HRESIMS were obtained on
a Bruker APEX-II mass spectrometer, and ESIMS were recorded
on an Agilent 1100-LC/MSD TrapSL mass spectrometer.
Silica gel GF254 (10–40 μm) prepared for TLC and silica gel
503.1769 [M − H] (calcd. for C22
H
31
O13, 503.1770).
2-Methyl-L-erythritol-1-O-(6-O-trans-sinapoyl)-β-D-glu-
copyranoside (2): yellow, amorphous powder; [α]18
−15.0
(c 0.50, CH OH); UV (CH OH) λmax (log ε): 213 (4.22), 240
D
3
3
(3.80), 300 sh (4.12) nm; IR (KBr) Vmax: 3398, 2936, 1700,
1635, 1602, 1515, 1458, 1424, 1382, 1283, 1154, 1118,
−
1
1
13
(
200–300 mesh) for column chromatography (CC) were
830 cm
;
H and C NMR data: see Table 1; HRESIMS m/z
−
obtained from Qingdao Marine Chemical Factory (Qingdao,
People's Republic of China). Octadecyl silica gel was purchased
from Merck Chemical Company Ltd. Macroporous resin D101
was a product of Chemical Plant of NanKai University (Tianjin,
China). Preparative HPLC separations were conducted using
a Shimadzu HPLC system equipped with a LC-6AD pump and
a SPD-20A detector using a C18 column (250 mm × 20 mm,
527.1725 [M + Na] (calcd. for C22
H
32
O13Na, 527.1735).
2.4. Acid hydrolysis of 1–2 and determination of the absolute
configuration of sugars
A solution of each compound (2.0 mg) in 2 M HCl (2 mL)
was stirred at 90 °C in a stoppered vial for 2 h. The solution after
5
μm; YMC Co. Ltd.). GC was carried out on an Agilent
GC-series system and performed with an HP-5 column
30 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA). All
2
cooling was evaporated under a stream of N . Anhydrous
pyridine solutions (1.0 mL) of each residue and L-cysteine
methyl ester hydrochloride (1.5 mg) were mixed and warmed
at 60 °C for 2 h. After drying the solution, trimethylsilyl
imidazole (150 μL) was added to the mixture, which was
warmed at 60 °C for another 1 h and then partitioned between
(
the reagents were of HPLC grade or analytical grade and
purchased from Tianjin Damao Chemical Company.
2
.2. Plant material
2
H O (500 μL) and cyclohexane (500 μL). The cyclohexane layer
was concentrated and analyzed by GC using an HP-5 column.
Temperatures of the injector and detector were 250 and 280 °C,
respectively. A temperature gradient system was used for the
oven, starting at 100 °C and increasing up to 140 °C at a rate
of 4 °C/min, and then increasing up to 170 °C for 8 min at a
rate of 13 °C/min, and finally, increasing up to 200 °C at a
rate of 5 °C/min. The peaks of authentic samples of D-glucose
and L-glucose after treatment in the same manner were
detected at 21.47 and 21.87 min.
The fruit of G. jasminoides was obtained from Jiangxi Province,
China, and identified by Professor Qishi Sun, of the School of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University. A voucher specimen (GJ-20091016) has been
deposited in the herbarium of the Department of Natural
Products Chemistry, Shenyang Pharmaceutical University.
2
.3. Extraction and isolation
Dried fruit (8.0 kg) of G. jasminoides was cut into pieces and
2.5. Acid hydrolysis of 1–2 and determination of the absolute
configuration of the aglycones of 1–2
extracted with 60% (v/v) EtOH (×3, 2 h each). The combined
extract (1000 g) was suspended in 3.0 L water and partitioned
with cyclohexane, EtOAc, and water-saturated n-butanol (×3,
A solution of compound 1 (17.0 mg) in 2 M HCl (17 mL)
was stirred at 90 °C in a stoppered vial for 2 h. The reaction
mixture was evaporated to dryness and then applied to an
open ODS column (2 × 5 cm, 50 μm). The column was eluted
3
.0 L each), successively. The EtOAc extract (70 g) was subjected
to silica gel column chromatography (CC) with a CHCl –MeOH
gradient solvent system (100:0 to 0:100) to obtain 9 fractions
E1–E9), which were combined according to TLC analysis.
3
(
2 2
with 50 mL H O and 50 mL MeOH, successively. The H O
Fraction E2 (10 g) was chromatographed on silica gel CC with a
cyclohexane–EtOAc gradient solvent system (100:0 to 0:100) to
obtain 3 (400 mg) and 4 (80 mg). Fraction E6 (5 g) was
eluate was concentrated and then purified by HPLC to
obtain the aglycone (1.8 mg). HPLC conditions were as
follows: Hypersil NH
solvent, MeCN–H O (85: 15); flow rate, 1 mL/min; column
temperature, 30 °C; detector, RID-10A. The aglycone was
detected at a t of 6.5 min.
2
column (4.6 × 250 mm, 10 μm);
separated by preparative HPLC eluted with MeOH–H
to afford 5 (12 mg, t 82.1 min), 6 (17 mg, t 109.4 min), 7
15 mg, t 130.0 min) and 8 (16 mg, t 180.0 min). Fraction E7
20 g) was chromatographed over silica gel eluted with CHCl
2
O (50:50)
2
R
R
(
R
R
R
(
3
–
A solution of compound 2 (6.5 mg) in 2 M HCl (6.5 mL)
was stirred at 90 °C in a stoppered vial for 2 h and then
treated in the same manner as compound 1 to obtain the
aglycone (0.8 mg). As we expected, these two aglycones
from 1 and 2 have the same chromatographic behavior and
spectral data.
MeOH (100:10 to 0:100) and resolution of fraction E74 (1 g) by
recrystallization with MeOH yielded 9 (200 mg).
The water extract (500 g) was chromatographed on D101
(
9
100 mesh) eluted with a gradient of EtOH–H
5:5) to obtain five fractions (W1–W5). Fraction W2 (2 g)
was subjected to ODS open CC eluted with MeOH–H O (5:95
to 50:50) and resolution of fraction W22 (300 mg) by
preparative HPLC (MeOH–H O, 40:60) afforded 1 (30 mg, t
6 min) and 2 (32 mg, t 69 min).
-Methyl-L-erythritol-4-O-(6-O-trans-sinapoyl)-β-D-glu-
2
O (0:100 to
2
2-Methyl-L-erythritol (the aglycone of 1 and 2): white
18
18
powder, [α]
MeOH); 1H NMR (D
2
D
−10.6 (c 0.085, H
2
O), [α]
D
−27.1 (c 0.07,
2
R
O, 600 MHz): δ 3.76 (1H, dd, J = 2.6,
5
R
11.6 Hz, H-4a), 3.59 (1H, dd, J = 2.6, 8.7 Hz, H-3), 3.53 (1H,
2
dd, J = 8.7, 11.6 Hz, H-4b), 3.51 (1H, d, J = 11.7 Hz, H-1a),
18
13
copyranoside (1): yellow, amorphous powder; [α]
D
−15.2
OH) λmax (log ε): 213 (4.20), 240
3.82), 300 sh (4.10) nm; IR (KBr) Vmax: 3396, 2934, 1702,
3.40 (1H, d, J = 11.7 Hz, H-1b), 1.10 (3H, s, 2-CH
(D
(C-4), 19.9 (2-CH
3
). C NMR
(
(
c 0.50, CH
3
OH); UV (CH
3
2
O, 150 MHz): 76.6 (C-2), 75.7 (C-3), 67.9 (C-1), 63.6
).
3

128
L. Yang et al. / Fitoterapia 89 (2013) 126–130
Table 1
1
H (300 MHz) and 13C NMR (75 MHz) data of compounds 1 and 2 in CD
3
OD.
No.
1
2
1
2
3
4
68.6
74.6
74.9
72.6
3.59 (o), 3.50 (o)
76.4
74.9
75.4
63.8
4.04 (d, 10.2), 3.47 (o)
3.88 (brd, J = 8.4)
4.24 (brd, J = 8.4), 3.66 (o)
3.78 (dd, J = 7.5, 3.0)
3.88 (dd, J = 11.1, 3.0)
3.64 (o)
5
19.9
1.18 (s)
19.4
1.16 (s)
1
2
3
4
5
6
′
′
′
′
′
′
105.1
75.4
77.9
71.8
75.6
64.8
4.43 (d, J = 7.2)
3.34 (o)
3.49 (o)
3.47 (o)
3.63 (o)
105.2
75.2
77.9
71.8
75.7
64.8
4.41 (d, J = 7.5)
3.35 (o)
3.45 (o)
3.47 (o)
3.63 (o)
4.58 (brd, J = 11.4)
4.60 (brd, J = 11.4)
4.40 (dd, J = 11.4, 6.0)
4
.40 (dd, J = 11.4, 6.0)
1
2
3
4
5
6
7
6
″
169.2
115.9
147.4
126.8
107.2
149.6
139.8
57.1
169.1
115.8
147.5
126.8
107.2
149.6
139.9
57.1
″
6.49 (d, J = 15.6)
7.70 (d, J = 15.6)
6.49 (d, J = 15.9)
7.71 (d, J = 15.9)
″
″
″, 9″
″, 8″
″
6.97 (s)
3.94 (s)
6.98 (s)
3.95 (s)
″,8″-OCH
3
All the signals were assigned by 1D and 2D NMR spectra.
2
.6. Cell culture and nitrite determination
Mouse monocyte–macrophage RAW 264.7 cells (ATCC
Experiments were performed in triplicate, and data are
expressed as the mean ± SD of three independent experiments.
TIB-71) were purchased from the Chinese Academy of Science.
RPMI 1640 medium, penicillin, streptomycin, and fetal bovine
serum were purchased from Invitrogen (New York). Lipopoly-
saccharide (LPS), dimethylsulfoxide (DMSO), 3-(4,5-dimethyl-
3. Results and discussion
Compound 1 was obtained as a yellow amorphous
powder with a molecular formula of C22H32O13 by analysis
of its HRESIMS spectrum. The 1H and C NMR spectra of 1
(Table 1) showed typical signals of a trans-double bond
[H-2″ (δ 6.49, 1H, d, J = 15.6 Hz), H-3″ (δ 7.70, 1H, d, J =
15.6 Hz), C-2″ (δ 115.9), C-3″ (δ 147.4)]. The presence of a
symmetrical 1,3,4,5-tetrasubstituted phenyl group was
deduced from the H-atom signals at δ 6.97 (2H, s, H-5″, 9″)
and the C-atom signals at δ 107.2 (C-5″, 9″), 126.8 (C-4″),
139.8 (C-7″), 149.6 (C-6″, 8″). Two methoxy groups are
located at C-6″ and C-8″ due to the HMBC correlations of δ
3.94 (6H, s)/C-6″, 8″. Furthermore, the HMBC correlations of
H-3″/C-4″, C-5″, C-9″, C-1″, and H-2″/C-1″, C-4″ revealed the
presence of a trans-sinapoyl moiety. The 13C NMR spectrum
showed 11 other carbon signals including one anomeric carbon
signal at δ 105.1 and one methyl group at δ 19.9. The remaining
nine carbon signals are in the range of δ 60–80 ppm, suggesting
a possible sugar and a possible polyalcohol moiety in the
molecule. Acid hydrolysis of 1 with 2 M HCl yielded D-glucose
(see Experimental section) [13]. The β-configuration was
established due to the coupling constant of the anomeric
13
2
-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), and
hydrocortisone were obtained from Sigma Co. (St. Louis, MO).
RAW 264.7 cells were suspended in RPMI 1640 medium
supplemented with penicillin (100 U/mL), streptomycin
(
100 μg/mL), and 10% heat-inactivated fetal bovine serum.
The cells were harvested with trypsin and diluted to a
suspension in fresh medium. The cells were seeded in
9
6-well plates with 1 × 105 cells/well and allowed to
2
adhere for 2 h at 37 °C in 5% CO in air. Then, the cells were
treated with 1 μg/mL of LPS for 24 h with or without various
concentrations of test compounds. DMSO was used as a
solvent for the test compounds, which were applied at a final
concentration of 0.2% (v/v) in cell culture supernatants. NO
production was determined by measuring the accumulation
of nitrite in the culture supernatant using Griess reagent
[
12]. Briefly, 100 μL of the supernatant from incubates was
mixed with an equal volume of Griess reagent (0.1%
N-[1-naphthyl]ethylenediamine and 1% sulfanilamide in 5%
H PO ). Cytotoxicity was determined by the MTT colorimet-
3 4
13
ric assay, after 24 h of incubation with test compounds. The
proton signal at δ 4.43 (1H, d, J = 7.2 Hz, H-1′) and the
C
−
concentration of NO
2
was calculated by a working line from
NMR data for the glucose were assigned as δ 105.1 (C-1′), 75.4
(C-2′), 77.9 (C-3′), 71.8 (C-4′), 75.6 (C-5′), 64.8 (C-6′) by
analysis of its HMQC and HMBC experiments. The polyalcohol
moiety was elucidated as 2-methyl-1,2,3,4-butanetetrol by
studying the remaining 1H and C NMR signals and by the
correlation from HMBC and HMQC spectra. Furthermore, the
HMBC correlations of H-6′ (δ 4.58, 4.40)/C-1″ and H-1′ (δ
4.43)/C-4 (δC 72.6), H-4 (δ 4.24, 3.66)/C-1′, suggesting that
0
, 1, 2, 5, 10, 20, 50, and 100 μM sodium nitrite solutions, and
the inhibitory rate on NO production induced by LPS was
−
calculated by the NO
2
levels as follows:
13
−
−
½
NO ꢁ −½NO ꢁ
2
LPS
2
LPSþsample
Inhibitory rate ð%Þ ¼ 100 ꢀ Â
Ã
Â
Ã
:
−
−
NO
− NO
2
LPS
2
untreated

L. Yang et al. / Fitoterapia 89 (2013) 126–130
129
3
Fig. 1. Structures of compounds 1–9.
the sinapoyl moiety was attached at C-6′ and the β-D-
glucopyranose was established at C-4, respectively. The
configuration of the aglycone was determined as (2R, 3S) by
comparing its NMR data and optical rotation with those
reported in the literature [14]. On the basis of the above
evidence, the structure of 1 was elucidated as 2-methyl-L-
erythritol-4-O-(6-O-trans-sinapoyl)-β-D-glucopyranoside
2-Methyl-1,2,3,4-butanetetrol has two stereocenters and
thus four stereoisomers: 2-methyl-D-erythritol, 2-methyl-L-
erythritol, 2-methyl-D-threitol and 2-methyl-L-threitol (Fig. 2).
These four stereoisomers, often being considered as SOA
(secondary organic aerosol) components in the atmosphere,
rarely exist in plants [15,16]. Only 2-methyl-D-erythritol and its
glycosides were isolated in a few references [11,18]. 2-Methyl-
D-erythritol, an important intermediate in the non-mevalonate
biosynthesis of terpenoids [17], was isolated from Torillis
japonica fruit [18], and its glycosides were obtained from
Pimpinella anisum L.[11]. However, no reports have been
published about 2-methyl-L-erythritol and its glycosides. In
the present study, two 2-methyl-L-erythritol glycosides (1 and
2) were isolated from the fruit of G. jasminoides. This is the first
report of 2-methyl-L-erythritol glycosides from plant. Based on
this finding, we propose that 2-methyl-L-erythritol might be a
new intermediate in the non-mevalonate biosynthesis of
terpenoids.
(
Fig. 1).
Compound 2 was obtained as a yellow amorphous powder
and had a molecular formula of C22 13 established by
32
H O
1
13
HRESIMS. The H and C NMR spectra of 2 were similar to
those of 1, showing the presence of a trans-sinapoyl moiety, a
2-methyl-1,2,3,4-butanetetrol moiety and a glucosyl moiety,
which was further elucidated as β-D-glucopyranose by the same
method as described for compound 1. In the HMBC spectrum,
correlations of H-6′ (δ 4.60, 4.40)/C-1″ (δ 169.1), H-1 (δ 4.04,
3.47)/C-1′ (δ 105.2), H-1′ (δ 4.41)/C-1 (δ 76.4), suggesting the
sinapoyl moiety was attached at C-6′ and the β-D-glucopyranose
was established at C-1, respectively. The configuration of the
aglycone of 2 was determined by using the same method as 1.
Thus, the structure of 2 was elucidated as 2-methyl-L-erythritol-
In addition, the known compounds were identified as
ursolic acid (3) [19,20], oleanolic acid (4) [21], methyl 3,4-
di-O-caffeoylquinate (5) [9], methyl 5-O-caffeoyl-3-O-
sinapoylquinate (6) [9], methyl 3,5-di-O-caffeoyl-4-O-(3-
1-O-(6-O-trans-sinapoyl)-β-D-glucopyranoside.
Fig. 2. The four stereoisomers of 2-methyl-1,2,3,4-butanetetrol.

130
L. Yang et al. / Fitoterapia 89 (2013) 126–130
Table 2
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a
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[
NO concentration of control group: 3.5 ± 0.2 μM. NO concentration of
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All compounds isolated from the fruit of G. jasminoides were
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by LPS in macrophages (Table 2). Cell viability was determined
by the MTT method to find whether inhibition of NO pro-
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shown in Table 2, hydrocortisone (IC50 64.3 ± 7.5 μM) was
used as positive control. Compounds 3 and 9 showed signifi-
cant inhibition effect with the IC50 values of 18.8 ± 1.1 μM and
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2
1.1 ± 1.5 μM, respectively. Compounds 5 and 7 exhibited
moderate activities, which were close to that of hydrocortisone.
In comparison with IC50 values of ursolic acid (3) and oleanolic
acid (4), we concluded compactly that the methyl group at
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-O-sinapoyl (8) had the best activities; when compound 6
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sinapoyl moiety in the quinic acid plays an important role in
defining the NO inhibitory effect.
In conclusion, the glycosides, triterpenoids, flavonoid and
quinic acid derivatives isolated from the fruit of G. jasminoides
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compounds 3 and 9 showed significant inhibition effect,
which provided candidates for the study of this medicinal
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Acknowledgments
The authors are grateful to Ms. Wen Li and Mr. Yi Sha of
the Department of Instrumental Analysis, Shenyang Pharma-
ceutical University, for measuring the NMR and mass spectra.