A new phenolic glycoside from the barks of Cinnamomum cassia

Article abstract of DOI:10.3390/molecules191117727

A new phenolic glycoside (1), named methyl 2-phenylpropanoate-2-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, was isolated from the barks of Cinnamomum cassia, along with three known phenolic glycosides and four known lignan glycosides. The structure of 1 was elucidated by extensive interpretation of spectroscopic data and chemical method. Selected compounds were evaluated for their immunosuppressive activities against murine lymphocytes. Compounds 1, 2, 6 and 8 exhibited differential inhibition against ConA-induced T cells proliferation.

Full text of DOI:10.3390/molecules191117727

Molecules 2014, 19, 17727-17734; doi:10.3390/molecules191117727
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A New Phenolic Glycoside from the Barks of Cinnamomum cassia
Junfen Zeng 2,3,†, Yongbo Xue , Yongji Lai , Guangmin Yao , Zengwei Luo ,
3,†
3
3
3
3
1,
Yonghui Zhang and Jinwen Zhang *
1
Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
2
Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan 430060, China
3
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation,
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, China
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: tjzhangjinwen@163.com;
Tel.: +86-27-8366-3253; Fax: +86-27-8369-2762.
External Editor: Derek J. McPhee
Received: 25 September 2014; in revised form: 29 October 2014 / Accepted: 29 October 2014 /
Published: 31 October 2014
Abstract: A new phenolic glycoside (1), named methyl 2-phenylpropanoate-2-O-β-D-
apiofuranosyl-(1→6)-O-β-D–glucopyranoside, was isolated from the barks of
Cinnamomum cassia, along with three known phenolic glycosides and four known lignan
glycosides. The structure of 1 was elucidated by extensive interpretation of spectroscopic
data and chemical method. Selected compounds were evaluated for their immunosuppressive
activities against murine lymphocytes. Compounds 1, 2, 6 and 8 exhibited differential
inhibition against ConA-induced T cells proliferation.
Keywords: Cinnamomum cassia; phenolic glycoside; immunosuppressive activities
1
. Introduction
The plant Cinnamomum cassia (Lauraceae), which originates in the south of China, is widely
cultivated in tropical or subtropical areas, such as Fujian, Guangdong, Guangxi, Yunnan and Hainan

Molecules 2014, 19
17728
Provinces of China, as well as Taiwan, India, Indonesia, Laos, Malysia, Thailand and Vietnam [1]. The
bark of C. cassia, well known as Cinnamomi cortex in China, has long been used as spice and
flavoring agents [2]. It also possesses various biological activities, such as insecticidal, anti-allergy,
antipyretic, anticancer, anti-Alzheimer′s disease, antidiabetic [3] and immunosuppressive activity [4].
In our previous study [5], three diterpenoids, cinncassiols F and G, and 16-O-β-D-glucopyranosyl-19-
deoxycinncassiol G, with unprecedented carbon skeletons, were isolated from the barks of C. cassia. It
is notable that two of them showed significant inhibitory effects on the proliferation of murine T cells
induced by ConA. Recently, with the aim of discovering more bioactive compounds from C. cassia,
the extracts of EtOAc and n-BuOH were further phytochemically studied, which led to the isolation of
a new phenolic glycoside (1), together with three known phenolic glycosides (2–4) and four known
lignan glycosides (5–8). The immunomodulatory activities against murine lymphocytes of the selected
compounds were reported in this work.
2
. Results and Discussion
The EtOAc extract of C. cassia was separated by various chromatographic techniques to yield a
new phenolic glycoside (1), along with seven known compounds. The known compounds were
identified as 3,4,5-trimethoxyphenol-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (2) [6],
cinnacasolide E (3) [7], phenol-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (4) [8], samwiside
(
5) [9], (6R,7R,8R)-7a-[(β-D-glucopyranosyl)oxy]lyoniresinol (6) [10], (6S,7R,8R)-7a-[(β-D-
glucopryanosyl)oxy]lyoniresinol (7) [10], and (6R,7S,8S)-7a-[(β-D-glucopyranosyl)oxy]lyoniresinol
(
8) [11], by comparison of their NMR data with those reported in literatures (Figure 1).
Figure 1. Structures of 1–8.
R2
6
R3
R1
O
5
1
H CO
3
6
OH
R4
4
2
3
5
7
OGlu
O
5''
4
''
1''
2''
O
6'
HO
8
HO
4'
H
O
HO
OCH3
'
3
''
HO
1
'
2
'
3
'
OH
OH OH
H CO
3
OCH3
O
OH
1
R =
1
OCH3
R = R = R = H
6
7
8
6R, 7S, 8S
6R, 7R, 8R
6S, 7R, 8S
2
3
4
2
3
4
R = H, R = R = R = OCH
1 2 3 4 3
R = R = OCH , R = R = H
1
3
3
2
4
R = R = R = R = H
1
2
3
4
OCH3
3
'
OCH3
4
'
O
6''
2'
O
2
4
'''
1'''
2'''
O
3
5
'''
4''
H
8'
1
HO
O
O
7
OH
HO
5''
8
3
'''
1'
5'
4
HO
2
''
1''
9'
7'
6'
3
''
OH
OH OH
6
5
9
HO
5

Molecules 2014, 19
17729
2
D
5
Compound 1 was isolated as amorphous powder, with [α]
−65.1 (c = 0.47, MeOH). Its molecular
+
formula, C21
H
30
O
12, was deduced from the HRESIMS peak at m/z 497.1616 [M+Na] (calcd for
−1
C
21
H O12Na, 497.1629). The IR spectrum showed the presence of carbonyl (1719 cm ) and hydroxyl
30
−1
1
group (3391 cm ). The H NMR spectrum of 1 (Table 1) showed proton signals for a
1
6
,2-disubstituted aromatic ring (δ
H
7.21, dd, J = 8.1, 1.2 Hz, H-3; 7.18, ddd, J = 8.1, 7.9, 1.9 Hz, H-4;
3.66, s).
.96, ddd, J = 7.9, 7.9, 1.2 Hz, H-5; and 7.17, dd, J = 7.9, 1.9 Hz, H-6), a methoxyl (δ
H
13C-NMR and DEPT spectra (Table 1) of 1 showed 21 carbon signals, including one methoxyl, one
ester carbonyl, five methylenes, eleven methines, and three quaternary carbons. Comparison of the
1
13
H- and C-NMR spectra with those of 4 revealed that 1 differed from 4 by the presence of a methyl
1
1
propionate at C-1. Thus, compound 1 was determined to be an analogue of 4. The HBMC and H- H
COSY spectra of 1 indicated the presence of a methyl propionate moiety, which was connected at C-1.
The HBMC correlations from H-1′ to C-2 and from H-1′′ to C-6′ revealed the linkages of the two
sugars to be the same as those of 4. The sugars were finally confirmed by comparing the retention
times of the three trimethylsilylthiazolidine derivatives obtained from GC analysis. On the basis of the
above evidences, compound 1 was determined and named methyl 2-phenylpropanoate-2-O-β-D-
apiofuranosyl-(1→6)-O-β-D-glucopyranoside.
1
13
Table 1. H-(400 MHz) and C-NMR (100 MHz) Data of 1 in CD OD.
3
Position
δ
H
δ
C
1
131.3
157.0
128.9
2
3
7.21 dd (8.1, 1.2)
4
7.18 ddd (8.1, 7.9, 1.9) 116.7
6.96 ddd (7.9, 7.9, 1.2) 123.5
5
6
7
7.17 dd (7.9, 1.9)
3.00 m
131.0
26.9
35.3
175.9
52.0
102.8
75.0
78.2
71.6
77.0
68.8
8
2.68 t (7.7)
9
OMe
3.66 s
4.88 d (7.8)
3.50 m
1
2
3
4
5
′
′
′
′
′
3.49 m
3.38 m
3.60 m
6
′a
′b
4.04 d (10.6)
3.64 m
6
1
2
3
′′
′′
′′
5.00 d (2.3)
3.93 d (2.3)
111.0
78.1
80.0
75.0
4
′′a
′′b
3.77 d (9.7)
3.97 d (9.7)
3.61 s
4
5
′′
65.6

Molecules 2014, 19
17730
The crude extract of C. cassia has been reported to exhibit potent anti-complement [12] and
immunosuppressive activities [4]. Thus, compounds 1, 2, and 5–8 were evaluated for their
immunomodulatory activities against murine lymphocytes. The results (Table 2) showed that
compounds 1, 2, 6, and 8 inhibited the proliferation of ConA-induced murine T cells at a concentration
of 50 μM, while the positive control at the concentration of 50 μM, resulted in an inhibitory ratio of
1
06.1% against T cells and an inhibitory ratio of 75.8% against B cells.
Table 2. Effects on Murine Lymphocyte Proliferation Induced by Concanavalin A (ConA)
(
5 μg/mL) or Lipopolysaccharide (LPS) (10 μg/mL) of the Selected Compounds 1, 2,
and 5–8 a.
Inhibitory/Enhanced Rate (%) b
ConA−Induced T Cell Proliferation LPS−Induced B Cell Proliferation
Concentration
μM)
Compounds
(
2
1
50
00
00
−56.7
−61.2
−35.4
−22.1
15.2
10.3
c25.2
25.6
30.1
7.8
1
2
5
6
7
8
2
5
1
2.5
2
1
50
2
00
00
−45.2
−38.8
−8.5
−12.5
−20.1
−27.9
−5.4
6.5
11.7
2.8
−47.7
−58.0
−23.6
−47.5
−11.3
−10.4
−13.8
2.5
18.7
35.1
−80.1 *
−58.3
−19.5
27.8
−35.1
−5.8
−12.5
10.3
6.2
28.4
22.2
10.7
−1.5
13.8
5
1
2.5
2
1
50
2
00
00
5
1
2.5
2
1
50
2
00
00
−13.2
20.5
41.4
5
36.1
1
2.5
−12.5
−22.7
78.5
62.4
58.4
2
1
50
2
00
00
5
1
2.5
12.5
2
1
50
2
00
00
−46.7
−10.3
25.6
5
57.8
1
2.5
14.5
52.2
2
1
50
2
00
00
−109.8 **
−106.0 **
−106.1 **
−97.1 **
−84.5 **
−106.1 **
−94.8 **
−75.8 *
−60.1
−54.5
CsA
5
1
2.5
a
Results are represented as mean ± SD based on three independent experiments. b -Inhibitory effect;
+stimulatory effect. (n = 3; * p < 0.05; ** p < 0.01 compared with control). Positive control: ConA or LPS;
negative control: DMSO.

Molecules 2014, 19
17731
3. Experimental
3.1. General Procedures
NMR spectra were recorded on a Bruker AM 400 spectrometer (Bruker, Ettlingcn, Germany), and
1
13
the H- and C-NMR chemical shifts were referenced to the solvent peaks for CD
49.15. HRESI-MS data were measured on a LC-LTQ-Orbitrap XL spectrometer (Thermo Fisher,
Waltham, MA, USA). Optical rotations were determined on a Perkin-Elmer PE-341LC polarimeter
3
OD at δ
H
3.31 and
δ
C
(PerkinElmer, Waltham, MA, USA). IR spectra were recorded on a Bruker VERTEX 70 spectrometer
(Bruker, Ettlingcn, Germany). UV spectra were recorded on a PerkinElmer Lambda 35 spectrophotometer
(PerkinElmer, Waltham, MA, USA). Semi-preparative HPLC was performed on an Agilent 1100
liquid chromatography (Agilent, Santa Clara, CA, USA) with an YMC (10 × 250 mm, 5 μm) column.
GC analysis was performed with a capillary column (30 m × 0.32 mm × 0.5 μm) on an Agilent 7890A
GC. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS (50 μm, YMC,
Kyoto, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Stockholm, Sweden) were used for
column chromatography.
3
.2. Plant Material
The barks of C. cassia were collected at Qujing, Yunan Province, China, in July 2010. The plant
material was identified by Prof. Changgong Zhang at the School of Pharmacy, Tongji Medical
College, Huazhong University of Science and Technology. A voucher specimen (No. 2010-0703) was
deposited in the herbarium of Hubei Key Laboratory of Natural Medicinal Chemistry and Resource
Evaluation, Tongji Medical College, Huazhong University of Science and Technology.
3.3. Extraction and Isolation
The air-dried stem barks of C. cassia (25 kg) were extracted with 95% ethanol at room temperature
three times. The EtOH extract was concentrated in vacuo (2.6 kg), and then suspended in water and
partitioned successively with petroleum ether, CHCl , EtOAc, and n-BuOH. The EtOAc soluble
extract was subjected to column chromatography (CC) (100–200 mesh) over silica gel, eluting with a
gradient of CHCl /MeOH to yield fractions 1–6. Fraction 3 (16 g) was chromatographed on YMC gel
column (ODS) eluted with MeOH/H O (10%–70%), and on silica gel with a gradient of CHCl /MeOH
from 25:1 to 10:1) to afford compounds 2 (25 mg), 3 (18 mg), and 4 (20 mg). Compound 1 (15 mg)
was isolated from fraction 4 and purified by semipreparative HPLC (MeOH/H O, 30:70). Fraction 5
21 g) was chromatographed on a silica gel CC with a gradient of CHCl /MeOH (15:1) to yield two
subfractions 5.1 and 5.2. Compound 5 (15 mg) was purified from fraction 5.1 by semipreparative HPLC
MeOH/H O, 20:80), compounds 6 (8 mg), 7 (5 mg), and 8 (10 mg) were isolated from fraction 5.2.
3
3
2
3
(
2
(
3
(
2
3
.4. Acid Hydrolysis
A solution of 1 (2.0 mg), in 2 M aqueous CF
a water bath [13]. The reaction mixture was diluted in H
2.0 mL × 3), then the aqueous layer was concentrated to remove CF
3
COOH (2.0 mL) was heated at 60 °C for 2 h in
O (2.0 mL) and extracted with EtOAc
COOH. The residue was
2
(
3

Molecules 2014, 19
17732
dissolved in pyridine (1.0 mL), to which L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine
1.0 mL) was added. Then, the mixture was kept at 60 °C for 30 min. After the reaction mixture was
dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.4 mL) at 60 °C for
0 min in a water bath. Finally, the mixture was partitioned between n-hexane and H O (1 mL each)
and the n-hexane extract was analyzed by gas chromatography (GC) under the following conditions:
column temperature, 250 °C; injection temperature, 250 °C; carrier N gas; flow rate 1.0 mL/min. In
(
3
2
2
the acid hydrolysate of 1, D-glucose and D-apiose were confirmed by comparison of the retention times
of their derivatives with those of D-glucose and D-apiose derivatives prepared in a similar way, which
showed retention times of 10.03 and 15.62 min.
Methyl 2-phenylpropanoate-2-O-β-D-apiofuranosyl-(1→6)-O-β-D–glucopyranoside (1): amorphous
2
5
powder; [α] −65.1 (c = 0.47, MeOH); UV (MeOH) λmax (log ε) = 213 (2.9) nm; IR (KBr) νmax 3391,
D
−
1 1
13
2
926, 2884, 1719, 1493, 1236, 1067, and 756 cm ; H-NMR (CD
3
OD, 400 MHz) and C-NMR (CD OD,
3
+
1
00 MHz) see Table 1; HRESIMS m/z: 497.1616 [M+Na] (calcd for C21
H O12Na, 497.1629).
30
3
.5. Lymphocyte Proliferation Test
Splenic lymphocytes were prepared as previously described by Kawaguchi et al. [14]. The
lymphocyte proliferation was determined by WST-8 assay using Cell Counting Kit-8 (Dojindo) [15].
6
The prepared spleen cells (2.5 × 10 cells) were seeded into each well of a 96-well microplate, and
various concentrations of compounds and 5 μg/mL of concanavalin A (Con A, from Canavaliaensiformis
Type III), for selective stimuli on T cells were added, cyclosporine A (CsA) was used as a positive
control. After being cultured at 37 °C with 5% CO for 48 h, 20 μL WST-8 was added to each well. The
2
absorbance at 450 nm with a 600 nm reference was detected on a microtiter plate reader (Bio-Rad 680).
All optical density (OD) values shown are the mean of triplicate sample ± SD. Controls with and
without ConA and LPS were used to establish the baseline proliferation for stimulated and unstimulated
cells. The lymphocyte proliferation rate was evaluated according to our reported procedure [5].
4
. Conclusions
A new phenolic glycoside, methyl 2-phenylpropanoate-2-O-β-D-apiofuranosyl-(1→6)-O-β-D-
glucopyranoside (1), was isolated from the barks of C. cassia, together with seven known compounds
2–8). The structure and relative configuration of the new isolate was elucidated by careful interpretation
(
of spectroscopic data. This is the first report of samwiside (5) in the genus of Cinnamomum.
Compounds 1, 2 and 5–8 were tested for their in vitro immunomodulatory activities. Compounds 2 and
6
showed weak inhibition against ConA-induced T cell proliferation at the dosage of 12.5–200 μM,
and the inhibitory effects are not dose-dependent. Interestingly, at a concentration of 200 μM,
compound 8 significantly inhibited ConA-induced T cell proliferation with an inhibition ratio of
80.1%, whilst at low concentrations of 25 and 12.5 μM, stimulated the proliferation of T cell.
Acknowledgments
We are grateful to Changgong Zhang at Huazhong University of Science and Technology for the
authentication of this plant material. This project was financially supported by the National Natural

Molecules 2014, 19
17733
Science Foundation of China (31370372, 81202423, and 31200258), Program for New Century
Excellent Talents in University, State Education Ministry of China (NCET-2008-0224).
Author Contributions
Jinwen Zhang and Yonghui Zhang designed the research; Junfen Zeng, Yongji Lai, Yongbo Xue,
Guangmin Yao, and Zengwei Luo performed research and analyzed the data; Jinwen Zhang and
Yonghui Zhang wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(