A new aromatic glucoside from stem bark of Illicium difengpi K.I.B. et K.I.M.

Article abstract of DOI:10.1080/14786419.2015.1004675

A new aromatic glucoside, namely 4-methoxyphenyl-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside (1), together with six known aromatic glucosides (2-7) were isolated from the stem bark of Illicium difengpi. The structures of these compounds were established by spectroscopic methods. The isolated aromatic glucosides were tested for anti-inflammatory activity. Compounds 1, 3 and 6 showed significant inhibitory effect on nuclear factor kappa B (NF-B) in RAW 264.7 macrophages induced by lipopolysaccharide.

Full text of DOI:10.1080/14786419.2015.1004675

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A new aromatic glucoside from stem
bark of Illicium difengpi K.I.B. et K.I.M.
ab
b
b
Chuntong Li , Zhijun Wu & Wansheng Chen
a
Department of Pharmacy and Pharmacology, First Affiliated
Hospital of PLA General Hospital, Beijing 100048, P.R. China
b
Department of Pharmacy, Changzheng Hospital, Second Military
Medical University, No. 415, Fengyang Rd, Shanghai 200003, P.R.
China
Published online: 03 Feb 2015.
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To cite this article: Chuntong Li, Zhijun Wu & Wansheng Chen (2015): A new aromatic glucoside
from stem bark of Illicium difengpi K.I.B. et K.I.M., Natural Product Research: Formerly Natural
Product Letters, DOI: 10.1080/14786419.2015.1004675
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2015.1004675
A new aromatic glucoside from stem bark of Illicium difengpi
K.I.B. et K.I.M.
a,b
Chuntong Li , Zhijun Wu * and Wansheng Chen *
b
b
a
Department of Pharmacy and Pharmacology, First Affiliated Hospital of PLA General Hospital, Beijing
b
00048, P.R. China; Department of Pharmacy, Changzheng Hospital, Second Military Medical
1
University, No. 415, Fengyang Rd, Shanghai 200003, P.R. China
(
Received 4 December 2014; final version received 3 January 2015)
OH
O
O
HO
OH
O
O
HO
O
O
O
O
OCH
OH
3
OH
OH
OCH
O
O
O
O
O
O
3
OH OH
OH
OH
OH OH
OH
OH
OH OHO HO H
OH
OH
R1
OCH
OH
OCH
OH
OCH3
3
3
3
R
R
3
OCH
2
O
1
2
3
R
R
R
=OCH , R =H
4
5 R =OCH
3 3
6 R =H
3
7
1
1
1
3
2
=R =H
2
=R =OCH
,
2
2
A new aromatic glucoside, namely 4-methoxyphenyl-(6-deoxy-a-L-mannopyranosyl)-
b-D-glucopyranoside (1), together with six known aromatic glucosides (2–7) were
isolated from the stem bark of Illicium difengpi. The structures of these compounds
were established by spectroscopic methods. The isolated aromatic glucosides were
tested for anti-inflammatory activity. Compounds 1, 3 and 6 showed significant
inhibitory effect on nuclear factor kappa B (NF-kB) in RAW 264.7 macrophages
induced by lipopolysaccharide.
Keywords: Illicium difengpi; anti-inflammatory; aromatic glucoside
1
. Introduction
Illicium difengpi is a small shrub growing in Guangxi province in China, which belongs to the
family Illiciaceae. The stem bark of I. difengpi was listed in Chinese Pharmacopoeia (Committee
2
010). It is an important traditional Chinese medicine and mainly used as a treatment of
rheumatic arthritics (RA). The alcoholature of stem bark of I. difengpi showed outstanding
clinical efficacy and pharmacodynamics potency. Previous studies led to the isolation of
phenylpropanoids, lignans, neolignans and triterpenoids from the extract of I. difengpi and
monoterpenoids and sesquiterpenes from volatility oil of its fruit (Wang et al. 1994; Yao 1996;
Huang et al. 1997; Fang et al. 2010; Chu et al. 2011; Li et al. 2013).
As part of an ongoing search for bioactive natural products from folk medicine, we have
conducted a phytochemical investigation on BuOH fraction of 80% aqueous EtOH extract of the
stem bark. This has led to the isolation of one new and six known aromatic glucosides, named as
4
-methoxyphenyl-(6-deoxy-a-L-mannopyranosyl)- b-D-glucopyranoside (1), phenyl-6-O-(6-
deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside (2), 3,4-dimethoxyphenyl-6-O-(6-deoxy-a-
L-mannopyranosyl)-b-D-glucopyranoside (3), 4-hydroxy-3,5-dimethoxyphenyl-b-D-glucopyr-
anoside (4), methyl ester-4-[[6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranosyl]oxy]-
3
,5-dimethoxybenzoic acid (5), 4-[[6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranosyl]
*
Corresponding authors. Email: wuzhijun999@sina.com; chenws126@126.com
q 2015 Taylor & Francis

2
C. Li et al.
oxy]-3-methoxy-benzoic acid (6) and 3,4-dimethoxyphenyl-6-O-D-apio-b-D-furanosyl-b-D-
glucopyranoside (7) (Figure 1). These compounds were evaluated for their anti-inflammatory
activity. This paper reports the isolation, structure elucidation and anti-inflammatory activity of
these aromatic glucosides.
2
. Results and discussion
The 80% ethanol extract of stem bark of I. difengpi was suspended in water and extracted
sequentially with petroleum ether, EtOAc and n-BuOH, respectively. The n-BuOH fraction was
subjected to column chromatography (CC) to yield compound 1 and six known aromatic
glucosides (2–7).
Compound 1 was obtained as white amorphous powder and its molecular formula was
2
determined to be C H O by HR-ESI-MS data (m/z 477.1620 [M þ COOH] , calc. for
1
9 28 11
1
C H O 432.1632), requiring six degrees of unsaturation. The C NMR and DEPT spectra of
3
1
9 28 11
1
methylenes, 1 methyl and 1 methoxyl. The C NMR spectrum showed particularly intense
exhibited 17 carbon resonances, classified into 2 quaternary carbons, 12 methines, 1
1
3
signals at d 119.2 and d 115.4 accounting for two magnetically equivalent carbons each (C-
C
C
3
þ C-5 and C-2 þ C-6). These aromatic carbons together with the quaternary carbons d 153.1
C
1
13
(C-1) and d 156.7 (C-4) gave evidence of a opposite-substituted benzene. The H and C NMR
C
spectra indicated that 1 contained a methoxyl group (d 3.70, 3H, s; d 56.0) connected to the
H
C
benzene group which was confirmed by HMBC correlation from OCH (d 3.70) to C-4 (d
C
3
H
1
56.7). Comparison of the C NMR spectrum of 1 with that of compound 2 (Feng et al. 2010)
3
1
indicated that the structure of the aglycone moieties of 1 was identical to those of compound 2.
0
In the HMBC experiment (Figure S1), the correlation from H-1 (d 4.67) to C-1 (d 153.1) was
C
H
observed, indicating that a glucopyranosyl group was attached to C-1 of the benzene. The
0
00
correlation from H-6 (d 3.55) to C-1 (d 102.2) indicated that the terminal mannopyranosyl
H
C
0
00
group was connected to the glucose unit by 6 ! 1 . The structure of 1 was established on the
1
13
basis of 1D and 2D NMR spectra ( H, C, DEPT, COSY, HSQC and HMBC). The absolute
configurations of glucopyranosyl and mannopyranosyl were further verified by acid hydrolysis
Figure 1. Structures of compounds 1–7.

Natural Product Research
3
and chiral GC-MS analysis. Therefore, the structure of 1 is assigned as 4-methoxyphenyl-(6-
deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside.
In addition to the new aromatic glucoside (1), six known aromatic glucosides phenyl-6-O-(6-
deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside (2) (Feng et al. 2010), 3,4-dimethoxyphenyl-
6
-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside (3) (Graikou et al. 2005), 4-hydroxy-
,5-dimethoxyphenyl-b-D-glucopyranoside (4) (Rojas et al. 2000), methyl ester-4-[[6-O-(6-
3
deoxy-a-L-mannopyranosyl)-b-D-glucopyranosyl]oxy]-3,5-dimethoxybenzoic acid (5) (Ma
et al. 2011), 4-[[6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranosyl]oxy]-3-methoxy-
benzoic acid (6) (Ma et al. 2011) and 3,4-dimethoxyphenyl-6-O-D-apio-b-D-furanosyl-b-D-
glucopyranoside (7) (Warashina et al. 2004) were isolated from I. difengpi. These compounds
were identified by spectral analysis and compared with spectroscopic data reported in the
literatures.
The anti-inflammatory activities of compounds 1–7 were determined by measuring
luciferase in RAW 264.7 cells stimulated with lipopolysaccharide (LPS) in vitro (Li et al. 2013;
Huang et al. 2014). Tripterygium tablets (TRT) and total glucosides of paenia (TGP) were used
as positive controls. The cytotoxic effects of tested compounds on LPS-stimulated RAW 264.7
cells were determined initially by MTT assay. The results showed that compounds 1–7 did not
affect cell viability at concentrations up to 25 mg/mL. As shown in Figure 2, the concentrations
of NF-kB in the RAW 264.7 cells pretreated with compounds 1, 3 and 6 at 10 mg/mL were
reduced by 64%, 59% and 46%, respectively, compared to LPS stimulated RAW 264.7 cells,
while the inhibitory rate (IR) of two positive controls TRT and TGP were 82% and 61%,
respectively (Figure 2). The results demonstrated that compounds 1, 3 and 6 showed a significant
inhibition of NF-kB production in LPS-stimulated RAW 264.7 cells (Figure 2).
3
3
. Experimental
.1. General experimental procedures
CC was performed on Sephadex LH-20 gel (40–70 mm, Amersham Pharmacia Biotech AB,
Uppsala, Sweden), YMC-GRL ODS-A (50 mm; YMC, MA, USA) and silica gel H (100–200 and
2
00–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). TLC analyses were
Figure 2. IR of NF-kB production (%) from LPS stimulated RAW 264.7 cells by compounds 1–7 at a
concentration of 10 mg/mL.

4
C. Li et al.
performed on Si 60 GF254 plates and visualised under UV light or by heating after spraying with
0% H SO /EtOH solution. 1D and 2D NMR spectra were recorded on an American Varian
1
2
4
Mercury Plus 400 NMR spectrometers (Agilent Technologies LDA UK Limited, Stockport,
Cheshire, UK). HR-ESI-MS were acquired on an Agilent 6220 TOF LC-MS (Agilent
Technologies Singapore (Sales) Pte Ltd, Singapore) instrument. GC-MS was conducted on an
Thermo Finnigan Trace GC (Thermo Technologies Co., San Jose, CA, USA) apparatus using an
L-Chirasil-Val column (25 m £ 0.32 mm).
3
.2. Plant material
The stem barks of I. difengpi were purchased from Caitongde Pharmacy, Shanghai, China, in
January 2010. Plant material was authenticated by Professor Lianna Sun, Department of
Pharmacognosy, School of Pharmacy, Second Military Medical University. A voucher specimen
(NO. 20100110) has been deposited in the Herbarium of the Department of Pharmacognosy,
School of Pharmacy, Second Military Medical University.
3
.3. Extraction and isolation
The air-dried stem bark of I. difengpi (40 kg) was powdered and extracted three times with 80%
ethanol under reflux. The solvent was concentrated to obtain a crude extract (1200 g), which was
suspended in water (10 L) and extracted with petroleum ether, EtOAc and BuOH, respectively.
The Fr 3 of BuOH section (40 g) was subjected to silica gel CC gradient with CH Cl /MeOH
2
2
(
eluted with CHCl /MeOH/H O (9:1:0.25) to afford compound 4 (1.0 mg). Fr (8 g) was
100:1 to 0:100) to give four subfractions (Fr3-1–Fr3-4). Fr3-1 (6 g) was subjected to silica gel CC
3
2
3-2
applied to ODS CC eluted gradient with MeOH/H O (50:50 to 100:0) to afford four subfractions
2
(
Fr3-2-1–Fr3-2-4). Fr3-2-3 (2.6 g) was purified by Sephadex LH-20 CC eluted with MeOH/H O
2
(
LH-20 eluted with MeOH/H O (50:50) to yield a mix of 2 and 3, which was separated with ODS
50:50) to yield compound 5 (28.9 mg) and 7 (18 mg). Fr3-3 (8.6 g) was subjected to Sephadex
2
CC eluted with MeOH/H O (50:50 to 75:25) to give 2 (34 mg) and 3 (34 mg). Fr (7.4 g) was
3-4
2
subjected to Sephadex LH-20 eluted with MeOH/H O (50:50) to afford four subfractions (Fr
2
3-4-
1–
7
Fr3-4-4), then Fr3-4-3 (2.2 g) was subjected to ODS CC eluted with MeOH/H O (40:60 to
2
5:25) to afford 6 (34.2 mg) and 1 (33.5 mg).
3
.4. Acid hydrolysis of compounds
Compound 1 was heated in 2 M HCl (1 mL) at 1208C for 1.0 h. The mixture was concentrated
and the residue was dissolved in 1-trimethylsilyl imidazole and pyridine (0.2 mL), and the
solution was stirred at 608C for 5 min. After drying the solution, the residue was partitioned
between CH Cl and H O (1 mL, 1:1 v/v). The organic phase was submitted to GC-MS analysis
2
2
2
using an L-Chirasil-Val column (0.32 mm £ 25 m). Temperatures of the injector and detector
were 2008C for both. A temperature gradient system was used for the oven, starting at 1008C for
min and increasing up to 1808C at a rate of 58C/min. Retention times for authentic samples
1
after being treated in the same manner with 1-trimethylsilyl imidazole in pyridine. D-glucose and
L-rhamnose were identified by comparing the retention time with those of authentic D-glucose
and L-rhamnose after treatment using the same method.
3
.5. Characterisation of compounds
4
-Methoxyphenyl-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside (1): amorphous pow-
1
2
5
der; ½aꢀ þ 8.9 (c ¼ 0.20, MeOH); H-NMR (CD OD, 400 MHz): d: 1.17 (3H, d, J ¼ 6.2 Hz, H-
D
3
00
0 0 00 0
6
), 3.30 (1H, m, H-5 ), 3.33 (1H, m, H-3 ), 3.36 (1H, m, H-4 ), 3.38 (1H, m, H-4 ), 3.46 (1H, m, H-

Natural Product Research
5
0
0
00
2
3
), 3.55 (1H, dd, J ¼ 11.0 and 6.6 Hz, H-6 a), 3.60 (1H, m, H-5 ), 3.65 (1H, dd, J ¼ 9.5 and
0
0
00
.5 Hz, H-2 ), 3.70 (3H, s, 4-OCH ), 3.79 (1H, dd, J ¼ 3.3 and 1.7 Hz, H-3 ), 3.96 (1H, d, J ¼ 11.0
3
0
00
0
and 1.6 Hz, H-6 b), 4.66 (1H, d, J ¼ 1.2 Hz, H-1 ), 4.67 (1H, d, J ¼ 7.2 Hz, H-1 ), 6.80 (2H, d,
1
3
J ¼ 9.6 Hz, H-2,6), 6.98 (2H, d, J ¼ 9.6 Hz, H-3,5); C-NMR (CD OD, 100 MHz): d: 153.1 (C-
3
0 0 0 0
1
7
6
), 115.4 (C-2,6), 119.2 (C-3,5), 156.7 (C-4), 103.4 (C-1 ), 74.0 (C-2 ), 76.8 (C-3 ), 71.6 (C-4 ),
8.0 (C-5 ), 67.9 (C-6 ), 102.2 (C-1 ), 72.4 (C-2 ), 72.2 (C-3 ), 74.9 (C-4 ), 69.8 (C-5 ), 18.0 (C-
0
0
00
00
00
00
00
00
2
), 56.0 (4-OCH ). HR-ESI-MS, m/z 477.1620 [M þ COOH] (calc. for C H O 432.1632).
3
19 28 11
4
. Conclusion
A new aromatic glucoside, 4-methoxyphenyl-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyrano-
side (1), together with six known aromatic glucosides (2–7) was obtained from the stem bark of
I. difengpi. Compounds 1, 3 and 6 showed significant inhibitory effect on nuclear factor kappa B
(NF-kB) in RAW 264. 7 macrophages induced by LPS with inhibitory rates of 64%, 59% and 46%,
respectively. The observed potential anti-inflammatory activity warrants further investigations.
Supplementary meterial
Supplementary data (Figure S1, 1D, 2D NMR spectra and HR-ESI-MS data of compound 1)
associated with this article are available online.
Funding
This work was supported by the National Natural Science Foundation of the People’s Republic of China
grant number 81274032], [grant number 81325024]; the Scientific Foundation of Shanghai [grant number
1DZ1971301].
[
1
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