Coumarins from Daphne feddei and their potential anti-inflammatory activities

Article abstract of DOI:10.1080/10286020.2011.621892

Chemical examination of the methanolic extract from the stem bark of Daphne feddei led to the isolation of three new dicoumarin glucosides (1-3), and eight known coumarins, dicoumarins and dicoumarin glucosides. Their structures were elucidated by extensive analysis of spectral data and comparison with the literature values. All compounds were tested for inhibitory activity against lipopolysaccharide-induced NO production in RAW 264.7 macrophages, and compounds 4 and 5 showed potent inhibitory activity with IC50 values of 0.161 and 0.127mM, respectively.

Full text of DOI:10.1080/10286020.2011.621892

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Coumarins from Daphne feddei and
their potential anti-inflammatory
activities
Shuang Liang a b , Yi Feng b , Jun-Mian Tian a , Min Lu c , Zhi Xiong b
& Wei-Dong Zhang a d
a School of Pharmacy, Shanghai Jiaotong University , Shanghai,
200240, China
b Shanghai University of Traditional Chinese Medicine Engineering
Research Center of Modern Preparation Technology of TCM,
Ministry of Education , Shanghai, 201203, China
c Department of Medical Informatics , Library of Second Military
Medical University , Shanghai, 200433, China
d Department of Natural Product Chemistry , School of Pharmacy,
Second Military Medical University , Shanghai, 200433, China
Published online: 25 Nov 2011.
To cite this article: Shuang Liang , Yi Feng , Jun-Mian Tian , Min Lu , Zhi Xiong & Wei-Dong Zhang
(2011) Coumarins from Daphne feddei and their potential anti-inflammatory activities, Journal of
Asian Natural Products Research, 13:12, 1074-1080, DOI: 10.1080/10286020.2011.621892
To link to this article: http://dx.doi.org/10.1080/10286020.2011.621892
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Journal of Asian Natural Products Research
Vol. 13, No. 12, December 2011, 1074–1080
Coumarins from Daphne feddei and their potential anti-inflammatory
activities
Shuang Liangab, Yi Fengb, Jun-Mian Tiana, Min Luc, Zhi Xiongb and Wei-Dong Zhangad*
b
aSchool of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China; Shanghai
University of Traditional Chinese Medicine Engineering Research Center of Modern Preparation
c
Technology of TCM, Ministry of Education, Shanghai 201203, China; Department of Medical
Informatics, Library of Second Military Medical University, Shanghai 200433, China; dDepartment
of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, Shanghai
200433, China
(Received 23 May 2011; final version received 6 September 2011)
Chemical examination of the methanolic extract from the stem bark of Daphne feddei
led to the isolation of three new dicoumarin glucosides (1–3), and eight known
coumarins, dicoumarins and dicoumarin glucosides. Their structures were elucidated
by extensive analysis of spectral data and comparison with the literature values. All
compounds were tested for inhibitory activity against lipopolysaccharide-induced NO
production in RAW 264.7 macrophages, and compounds 4 and 5 showed potent
inhibitory activity with IC50 values of 0.161 and 0.127 mM, respectively.
Keywords: Daphne feddei; coumarin; RAW 264.7 macrophages; nitric oxide
1. Introduction
Daphne feddei Levl., a common ever-
green shrub in Southwest China, is a rich
resource of coumarins from Thymelaea-
ceae, and its stem bark is used in folk
medicine for the treatment of injuries such
as falls and bruises [5]. A detailed
investigation on the title plant resulted in
the isolation of three new dicoumarin
glucosides (1–3; Figure 1), together with
eight known coumarins: 7-hydroxycou-
marin (4) [6], daphnetin (5) [7], skimmin
(6) [8], daphnin (7) [9], daphnetin-8-O-b-
glucopyranoside (8) [9], giraldoid A (9)
Coumarins are secondary metabolites
widely distributed in the plant kingdom,
and especially abundant in the families of
Umbelliferae, Fabaceae, Asteraceae, and
Thymelaeaceae [1]. In the past years,
research efforts on the coumarins focused
on isolation and identification of new
compounds, biosynthetic patterns, and
their potential biological actions. Modern
pharmaceutical studies have proved that
coumarins and their derivatives have a
wide range of bioactivities [2–4], which is
consistent with the fact that plants rich in
coumarins have long been used as
medicinal herbs in different cultures.
Recently, the search for lead compounds
in drug discovery from medicinal plants
has led to a resurgence of interest in
coumarins.
[10],
7,70-dihydroxy-6,80-bicoumarin
(bicoumarin) (10) [11], and daphgilin-7-
O-b-glucopyranoside (daphnolin) (11)
[12]. This paper describes the structural
elucidation of compounds 1–3, and the
inhibitory activity of the isolated com-
pounds against NO production induced by
lipopolysaccharide (LPS) in macrophages.
*Corresponding author. Email: wdzhangy@hotmail.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.621892
http://www.tandfonline.com

Journal of Asian Natural Products Research
1075
4'a
8'a
4'a
OH
O
O
HO
O
8'a
O
O
O
1''
HO
HO
4a
OH
3
4a
6
OH
3
6
O
8a
HO
HO
O
O
1''
O
8a
O
O
HO
OH
1
2
4'
4'a
4a
OH
O
7'
O
HO
HO
1''
O
O
O
OH
O
4
O
HO
OH
3
Figure 1. Chemical structures of compounds 1–3.
2. Results and discussion
H-100/C-70 (d ¼ 158.3) suggested that
the sugar moiety was attached to C-70
(Figure 2). In fact, compared with the
known bicoumol [11], the NMR spectral
data of 1 showed a great similarity to the
known compound except for the additional
signals due to a b-glucosyl group. Thus,
compound 1 was deduced as 7-hydroxy-70-
(b-glucopyranosyloxy)-(6,80-bi-2H-1-ben-
zopyran)-2,20-dione, and named bicoumol-
70-O-b-glucopyranoside.
Compound 2 was obtained as a white
powder. The molecular formula C24H20O11
was established by HR-ESI-MS, which is
the same as that of 1. Its 1H and 13C NMR
spectral data almost resembled those of 1,
suggesting that it is an isomer of 1.
The HMBC correlation of H-100/C-7
(d ¼ 158.8) revealed that the sugar moiety
was attached to C-7, rather than C-70
(Figure 2). Thus, compound 2 was deduced
as 7-(b-glucopyranosyloxy)-70-hydroxy-
(6,80-bi-2H-1-benzopyran)-2,20-dione, and
named bicoumol-7-O-b-glucopyranoside.
Compounds 1 and 2 changed into each
other in the MeOH–H2O solution within
24 h (Figure 3), and acid hydrolysis of
them afforded glucose and corresponding
Compound 1 was obtained as a white
powder. The molecular formula
C24H20O11 was determined by HR-ESI-
MS. The 13C NMR (Table 1) and DEPT
spectra of 1 showed 24 carbon resonances
ascribed to a methylene, 13 methines, and
1
10 quaternary C atoms. In the H NMR
spectrum, two doublets with an AB
coupling pattern (d ¼ 6.38, d, J ¼ 9.6 Hz,
H-3; d ¼ 8.03, d, J ¼ 9.6 Hz, H-4), along
with two singlets (d ¼ 7.58, H-5;
d ¼ 7.31, H-8), were indicative of a 6,7-
disubstituted coumarin moiety. Two pairs
of doublets with an AB coupling pattern
(d ¼ 6.15, d, J ¼ 9.6 Hz, H-30; d ¼ 7.92,
d, J ¼ 9.6 Hz, H-40; d ¼ 7.54, d,
J ¼ 8.4 Hz, H-50; d ¼ 6.94, d, J ¼ 8.4 Hz,
H-60) indicated the existence of a 7,8-
disubstituted coumarin moiety. The 13C
NMR spectrum suggested that 1 contained
a glucose unit (d 100.3, 77.1, 76.6, 73.0.
69.6, and 60.7). The anomeric H-100 of
the glucose moiety was determined to be
b-oriented on the basis of the coupling
constant for H-100 (d ¼ 4.99, d,
J ¼ 7.2 Hz). The HMBC correlation of

1076
S. Liang et al.
Table 1. 13C and 1H NMR spectral data of compounds 1–3 (d in ppm, J in Hz).
1a 2a
3b
d(C)
d(H)
d(C)
d H)
d(C)
d(H)
2
3
4
4a
5
6
7
8
8a
20
30
40
40a
50
60
70
800
800a
100
2
160.2
113.1
144.1
111.5
131.2
119.0
158.5
102.9
153.1
160.4
112.3
144.2
111.3
128.5
112.9
158.3
111.2
154.7
100.3
73.0
160.2
113.2
144.0
111.4
131.4
119.6
158.8
102.6
152.1
160.4
112.3
144.8
111.3
128.8
112.8
158.0
111.1
154.7
100.1
73.1
161.1
113.8
139.3
108.1
145.5
104.4
150.6
133.6
145.2
161.1
115.2
144.9
116.8
115.6
118.5
149.0
138.0
145.5
103.2
75.1
6.38 (d, 9.6)
8.03 (d, 9.6)
6.38 (d, 9.6)
8.03 (d, 9.6)
6.36 (d, 9.6)
8.04 (d, 9.6)
7.58 (s)
7.31 (s)
7.60 (s)
7.30 (s)
7.42 (s)
6.15 (d, 9.6)
7.92 (d, 9.6)
6.21 (d, 9.6)
8.00 (d, 9.6)
6.43 (d, 9.6)
7.71 (d, 9.6)
7.54 (d, 8.4)
6.94 (d, 8.4)
7.58 (d, 8.4)
6.98 (d, 8.4)
6.96 (d, 8.4)
7.03 (d, 8.4)
4.99 (d, 7.2)
2.92 (m)
3.23 (m)
3.05 (m)
3.39 (m)
3.39 (m), 3.74 (m)
5.09 (d, 7.8)
2.93 (m)
3.23 (m)
3.07 (m)
3.39 (m)
3.43 (m), 3.79 (m)
5.82 (d, 6.6)
4.28–4.40 (m)
3.99 (m)
4.28–4.40 (m)
4.28–4.40 (m)
4.28–4.40 (m)
300
400
500
600
76.6
69.6
77.1
60.7
76.3
69.4
77.0
60.5
79.5
71.4
78.8
62.5
a Measured in DMSO-d6.
b Measured in pyrindine-d5.
aglycones, of which NMR spectral data
were in accordance with the respective data
reported before [11]. These pieces of
evidence further confirmed their structures.
Compound 3 was obtained as a white
powder. Its HR-ESI-MS gave a pseudo-
molecular ion peak at m/z 539.0836
[M þ Na]þ, corresponding to the molecu-
lar formula of C24H20O13. Its 1H NMR
spectrum displayed signals of two pairs of
doublets with an AB coupling pattern
(d ¼ 6.43, d, J ¼ 9.6 Hz, H-30; d ¼ 7.71,
d, J ¼ 9.6 Hz, H-40; d ¼ 6.96, d, J ¼ 8.4
Hz, H-50; d ¼ 7.03, d, J ¼ 8.4 Hz, H-60),
indicating the existence of a 7,8-dioxyge-
nated coumarin moiety, and signals of two
doublets with an AB coupling pattern
(d ¼ 6.36, d, J ¼ 9.6 Hz, H-3 and
d ¼ 8.04, d, J ¼ 9.6 Hz, H-4) and a singlet
(d ¼ 7.42, H-6), indicative of a trioxyge-
nated coumarin moiety. The 13C NMR and
DEPT spectra revealed 24 carbon reson-
ances of a methylene, 12 methines, and 11
quaternary C atoms including those for a
glucose unit (d ¼ 103.2, 79.5, 78.8, 75.1.
71.4, and 62.5). The anomeric H-100 of
the glucose moiety was determined to
be b-oriented on the basis of its coupling
constant (d ¼ 5.82, d, J ¼ 6.6 Hz).
The HMBC correlation of H-100/C-70
(d ¼ 149.0) further suggested that the
sugar moiety was attached to C-70
(Figure 2). In fact, the NMR spectra of 3
were very similar to those of daphgilin
[13] except for the additional signals due
to a b-glucose group. Thus, compound 3
was deduced as 70-(b-glucopyranosyloxy)-
5-[(2-oxo-7,8-dihydroxy-2H-1-benzopyr
an-8-yl)oxy]-2H-1-benzopyran-2-one, and
named daphgilin-70-O-b-glucopyranoside.
Similar to compounds 1 and 2, compounds
3 and daphgilin-7-O-b-glucopyranoside

Journal of Asian Natural Products Research
1077
H
H
O
H
H
O
H
O
H
H
O
H
O
OH
O
HO
H
H
HO
HO
OH
OH
3
H
O
O
HO
HO
O
O
HO
O
O
H
OH
H
1
2
H
H
OH
H
O
H
O
O
HO
HO
O
O
OH
O
H
O
HO
OH
3
Figure 2. Key HMBC (H ! C) and NOESY (H $ H) correlations of compounds 1–3.
(11) changed into each other in MeOH–
H2O solution slowly within a month.
of NO with IC50 values of 0.161 and
0.127 mM, respectively.
All 11 isolates were tested for
inhibitory activity against LPS-induced
NO production in RAW 264.7 macro-
phages. Compounds 4 and 5 showed
inhibitory activity against the production
Since nitric oxide (NO) plays an
important role in the inflammatory process
[14], inhibitors of NO release may be
considered as potential therapeutic agents
in inflammatory diseases [15]. Our
OH
O
O
HO
O
O
O
HO
HO
O
OH
OH
O
O
HO
HO
O
O
-
O
HO
O
OH
1
2
H2O
-
MeOH
rt, 24h
rt, 24h
MeO + H
H2O
MeOH
MeO + H
O
O
O
OH
H
O
HO
HO
O
O
O
OH
OMe
Figure 3. Presumed mechanism of compounds 1 and 2. (There are two SN2 conversions, and
absorption configuration of sugar is maintained.)

1078
S. Liang et al.
investigation showed that compounds 4
and 5 may represent potential nitric oxide
synthase inhibitors.
China, and identified as D. feddei Levl. by
Prof. Li-Shan Xie of the Kunming Institute
of Botany. A voucher specimen (No.
200607-12) has been deposited in the
Herbarium of the School of Pharmacy,
Second Military Medical University.
3. Experimental
3.1 General experimental procedures
Optical rotations were acquired with a
Perkin-Elmer 341 polarimeter (Perkin-
Elmer, Beaconsfield, UK), whereas UV
spectra were obtained by using a Shimadzu
UV-2550 UV–vis spectrophotometer (Shi-
madzu, Kyoto, Japan). IR spectra were
recorded on a Bruker Vector 22 spec-
trometer (Bruker Optik GmbH, Ettlingen,
Germany) with KBr pellets. NMR spectra
were recorded on a Bruker Avance 400
NMR spectrometer (Bruker BioSpin
GmBH, Rheinstetten, Germany) with
TMS as an interal standard. HR-ESI-MS
were measured using a Q-TOF micro mass
spectrometer (Waters, Millford, MA,
USA). Materials for column chromatog-
raphy were silica gel (100–200 mesh;
Huiyou Silica Gel Development Co. Ltd,
Yantai, China), silica gel H (10–40 mm;
Yantai), Sephadex LH-20 (40–70 mm;
Amersham Pharmacia Biotech AB,
Uppsala, Sweden), and YMC-gel ODS-A
(50 mm; YMC, Allentown, PA, USA).
Preparative TLC (0.4–0.5 mm) was con-
ducted with glass precoated silica gel GF254
plates (Yantai). Aminoguanidine, LPS,
sulphanilamide, and naphthylethylenedia-
mine were purchased from Sigma-Aldrich
(St Louis, MO, USA). RAW 264.7 murine
macrophages were obtained from the
Shanghai Institute of Cell Biology, Chinese
Academy of Sciences (Shanghai, China)
and maintained in media recommended by
the suppliers, supplemented with 10% fetal
bovine serum (Gibco, Paisley, UK), peni-
cillin (100 U ml21), and streptomycin
(100 mg ml21) in a humidified 5% CO2
atmosphere at 378C.
3.3 Extraction and isolation
The air-dried and powdered stem bark of
D. feddei (6.5 kg) was percolated with
methanol for 4 h at room temperature for
three times. The solvent was evaporated
under reduced pressure. Then, the extract
was suspended in H2O and partitioned
with petroleum ether, EtOAc, and BuOH,
successively. The EtOAc extract (400 g)
was subjected to column chromatography
on silica gel (200–300 mesh, 1000 g),
eluted successively with gradient CHCl3–
MeOH mixtures of increasing polarity and
separated into 18 fractions (F1– F18).
F5 (35 g) was chromatographed on silica
gel with CHCl3–MeOH (100:0, 100:1) to
give compounds 4 (200 mg) and 5 (10 g).
F11 (3.5 g) was chromatographed on silica
gel with CHCl3 –MeOH (50:1, 25:1)
followed by Sephadex LH-20 with
MeOH to give compounds 6 (30 mg),
7 (10 mg), 8 (200 mg), and 9 (400 mg). F15
(3.5 g) was chromatographed on silica gel
with CHCl3–MeOH (10:1) and separated
into five sub-fractions (F15-1–F15-5). F15-2
was re-purified by preparative thin layer
chromatography (PTLC), eluting with
CHCl3 –MeOH (20:1) to afford com-
pounds 1 (10 mg) and 2 (20 mg). F15-4
was further purified by column chroma-
tography over octadecylsilan (MeOH–
H2O 30:70, 40:60), affording compounds
3 (400 mg), 10 (150 mg), and 11 (50 mg).
3.3.1 Bicoumol-70-O-b-glucopyranoside
(1)
A white powder (MeOH); TLC (silica gel,
CHCl3 –MeOH,
18
3.2 Plant Material
10:1):
½aꢀD : þ 9 (c 0.17, MeOH); UV (MeOH):
Rf ¼ 0.62;
The plant material was collected in July
2006 in Kunming City, Yunnan Province,
lmax (log 1) 317 (3.29); IR (KBr): nmax

Journal of Asian Natural Products Research
1079
3401, 2917, 1708, 1602, 1560, 1498, 1381,
1231, 1037, 829, and 618 cm21; For
1H and 13C NMR spectral data, see
Table 1; HR-ESI-MS: m/z 507.0906
[M þ Na]þ (calculated for C24H20O11Na,
507.0903).
3.3.5 Assay for inhibition ability against
LPS-induced NO production
RAW 264.7 macrophages were seeded in
24-well plates (105 cells per well). The
cells were pretreated with compounds at
different concentrations, aminoguanidine
or vehicle solution for 20 min, and then,
incubated with LPS (1 mg ml21) for 24 h.
The amount of NO was assessed by
determining the nitrite concentration in
the cultured RAW 264.7 macrophage
supernatants with Griess reagent. One
hundred microliters of supernatants were
incubated, in sequence, with 50 ml 1%
sulphanilamide and 50 ml 0.1% naphthy-
lethylenediamine in 2.5% of phosphoric
acid solution. The absorbances at 570 nm
were read using an absorbance microplate
reader Elx800 (BioTek Instruments, Inc.,
Winooski, VT, USA) [16].
3.3.2 Bicoumol-70-O-b-glucopyranoside
(2)
A white powder (MeOH); TLC (silica gel,
17
CHCl3-MeOH, 10:1): Rf ¼ 0.50; ½aꢀD : þ 5
(c 0.14, MeOH); UV (MeOH): lmax (log 1)
317 (3.31); IR (KBr): nmax 3411, 2920, 1707,
1602, 1498, 1383, 1312, 1231, 1163, 1078,
1
901, 829, and 618 cm21; For H and 13C
NMR spectral data, see Table 1; HR-ESI-
MS: m/z 507.0909 [M þ Na]þ (calculated
for C24H20O11Na, 507.0903).
3.3.3 Daphgilin-70-O-b-glucopyranoside
(3)
Acknowledgements
A white powder (MeOH); TLC (silica gel,
18
This work was financially supported by
program
CHCl3–MeOH, 5:1): Rf ¼ 0.46; ½aꢀD :
NCET
Foundation,
NSFC
(30725045), the Special Program for New
Drug Innovation of the Ministry of Science and
Technology, China (2009ZX09311-001,
2008ZX09308-005), Shanghai Leading Aca-
demic Discipline Project (B906) and in part by
the Scientific Foundation of Shanghai, China
(09DZ1975700, 09DZ1971500).
275 (c ¼ 0.10, MeOH); UV (MeOH):
lmax (log 1) 259 (3.42), 313 (3.47); IR
(KBr): nmax 3421, 2924, 1707, 1619, 1575,
1491, 1384, 1339, 1267, 1166, 1077, 826,
and 609 cm21; For 1H and 13C NMR
spectral data, see Table 1; HR-ESI-MS:
m/z 539.0836 [M þ Na]þ (calculated for
C24H20O13 Na, 539.0802).
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