Alkaloids and phenolic glycosides from Clematis mandshurica and their inhibitory effects against NO production in LPS-induced RAW 246.7 macrophages

Article abstract of DOI:10.1016/j.phytol.2016.08.005

Two new alkaloids (1 and 2), one new phenolic glycoside (3), and five known structures (4–8) were isolated from the roots and rhizomes of Clematis mandshurica. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products

Full text of DOI:10.1016/j.phytol.2016.08.005

Phytochemistry Letters 17 (2016) 238–241
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Alkaloids and phenolic glycosides from Clematis mandshurica and their
inhibitory effects against NO production in LPS-induced RAW 246.7
macrophages
a
a
a
a
b
b,
Qiang Fu , Jiang Chen , Hai-Mei Yuan , Yu Ma , Tian Yu , Liang Zou *
a
School of Pharmacy and Bioengineering, Chengdu University, Chengdu 610106, China
School of Medicine and Nursing, Chengdu University, Chengdu 610106, China
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 20 May 2016
Received in revised form 5 August 2016
Accepted 8 August 2016
Available online xxx
Two new alkaloids (1 and 2), one new phenolic glycoside (3), and five known structures (4–8) were
isolated from the roots and rhizomes of Clematis mandshurica. Their structures were elucidated on the
basis of spectroscopic evidence and hydrolysis products. All isolates were evaluated for inhibitory activity
against LPS-induced nitric oxide (NO) production in RAW 246.7 macrophages. Feruloyl and isoferuloyl
derivates showed moderate inhibitory effects with the inhibition ratios (%) from 29.3 to 38.7 at 50
whereas 3-carboxy-indole derivates showed potent inhibitory activities with IC50 from 7.7 to 12.4
m
m
M,
M.
Keywords:
Clematis mandshurica
Alkaloids
ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phenolic glycosides
Anti-inflammatory activity
Nitric oxide production
1. Introduction
and structure elucidation, and anti-inflammatory activity of these
compounds.
Clematis mandshurica Rupr. (Ranunculaceae) is widely distrib-
uted in northern China. Its roots and rhizomes have been used as a
traditional Chinese medicine “Weilingxian”, which is prescribed
for treatment of inflammation, rheumatoid arthritis and tumor.
2. Results and discussion
The EtOH extract of dried roots and rhizomes of C. mandshurica
was subjected to silica gel column chromatography and prep-HPLC
to obtain three new compounds (1–3), together with five known
(
Chawala et al., 2012). Up to now, many chemical components have
been isolated from this plant, including triterpene saponins (Shi
et al., 2006a), alkaloids (Shi et al., 2006b), and phenolic glycosides
structures, (4) 7-O-
nosyl 3-indolecarbonate (Shi et al., 2006b), (5)
anosyl-(1 !6)- -glucopyranosyl 3-indolecarbonate (Shi et al.,
2006b), (6) 5-O-feruloyl-2-deoxy- -ribono- -lactone (Shi et al.,
2006c), (7) 5-O-isoferuloyl-2-deoxy- -ribono- -lactone (Dilshara
et al., 2015), (8) Ibotanolide B (Lee et al., 2008).
a
-L-rhamnopyranosyl-(1! 6)-
b
-
D
-glucopyra-
(
Shi et al., 2006c). Pharmacological studies showed that the anti-
a-L-rhamnopyr-
inflammatory and anti-tumor biological effects of this plant may
be attributed to the triterpene saponins (Lee et al., 2007; Li et al.,
b-D
D
g
2013). However, bioactivity of alkaloids and phenolic glycosides
D
g
from this plant has not been well investigated. Previously, we
reported on the isolation and structural elucidation of 27
triterpene saponins from C. chinensis and their inhibitory activity
against COX-1/COX-2 enzymes (Fu et al., 2010). In our continuing
effort to seek for the bioactive components from genus clematis, a
re-investigation on the roots and rhizomes of C. mandshurica led to
the discovery of two new alkaloids, one new phenolic glycoside
Their structures were elucidated on the basis of spectroscopic
evidence and hydrolysis products. Compounds 1–8 were screened
for inhibitory effects against nitric oxide production in LPS-
induced RAW 246.7 macrophages.
Compound 1 was isolated as white amorphous powder. The
HRESIMS (negatve-ion mode) experiment reveled a pseudo-
ꢀ
(
Fig. 1), and five known structures. Herein, we report the isolation
molecular ion peak [MꢀH] at m/z 274.0711, in agreement with
5
the molecular formula C14H13NO . Its IR spectrum showed the
ꢀ
1
absorption of ester carbonyl group at 1705 cm and aromatic ring
ꢀ
1
1
absorptions at 1632 and 1520 cm . In the H NMR spectrum of 1,
four aromatic signal peaks were assigned to H-4 ( 8.10, m), H-5 (
7.21, m), H-6 ( 7.22, m), and H-7 ( 7.47, m), which suggested the
d
d
*
Corresponding author.
E-mail address: zouliang@cdu.edu.cn (L. Zou).
d
d
http://dx.doi.org/10.1016/j.phytol.2016.08.005
874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1

Q. Fu et al. / Phytochemistry Letters 17 (2016) 238–241
239
Fig. 1. The structures of compounds 1–3.
existence of the ortho-disubstituted benzene ring. The broad
singlet signal at 8.05 was characteristic of the H-2 olefinic proton
glucose and L for rhamnose by GC analysis of chiral derivatives of
the monosaccharides in the hydrolysate of 3 (see experimental
section). The relatively large coupling constants (8.0 Hz) for the
anomeric protons in the H NMR spectra of 3 suggested that the
glucopyranosyl moiety has a b-configuration. The a-configuration
d
1
3
in indole. The C NMR spectrum displayed 14 carbons, including
eight aromatic carbons, two carbonyl carbon, one oxygenated
methylene carbon, two oxygenated methine carbons, and one
methylene carbon. Comparison of the 1H and C NMR spectral
1
13
of the rhamnopyranosyl moiety was determined from the broad
singlet observed for the anomeric proton. In the HMBC spectrum,
data for 1 with the known compound
1 !6)- -glucopyranosyl 3-indolecarbonate (5) (Shi et al.,
006b) revealed that the two compounds have same skeleton,
a-L-rhamnopyranosyl-
0
00
(
b
-D
H
the anomeric proton signals at d 5.58 (H-1 ) and 4.72 (H-1 )
2
showed cross-peaks with the carbon signals at
d
C
168.5 (C-9) and
0
suggesting that 1 was a 3-carboxy-indole derivate. This deduction
was confirmed by HMQC correlations between H-2/C-2, H-4/C-4,
H-5/C-5, H-6/C-6, and H-7/C-7, and HMBC correlations between H-
67.8 (C-6 ), respectively. These signals provide evidence to
determine the linkages between the sugars, and the sugar and
the aglycone. From the above evidence, the structure of 3 was
4
/C-8, H-7/C-9, and H-2/C-10.
In addition, signals due to a hydroxylated
established as 9-O-
nosyl isoferuloyl ester.
All isolates were evaluated for inhibitory activity against LPS-
induced nitric oxide (NO) production in RAW 246.7 macrophages
(see Experimental). Dexamethasone (Sigma, St. Louis, MO, USA)
a
-
L
-rhamnopyranosyl-(1 !6)-
b-D-glucopyra-
g-lactone were
0
0
observed at
d
H
2.51 (dd, J = 3.5, 18.0 Hz, H-2 ), 3.02 (dd, J = 7.0,
0
0
18.0 Hz, H-2 ), 4.44 (m, H-3 ), 4.58 (m, H-4 ), 4.22 (dd, J = 5.0,
0
0
0
1
3
2.0 Hz, H-5 ), and 4.42 (dd, J = 3.0, 12.0 Hz, H-5 );
d
C
177.6 (C-1 ),
8.5 (C-2 ), 69.6 (C-3 ), 87.0 (C-4 ), and 64.4 (C-5 ). The structure of
-lactone was identified as 2-deoxy- -ribono-
-lactone by comparison of its spectral data with literature values
Dilshara et al., 2015; Shi et al., 2006c). In the NOESY spectrum, no
0
0
0
0
was used as positive control with IC50 values of 0.9 ꢁ 0.11
mM
hydroxylated
g
(
g
D
(n = 3). Feruloyl and isoferuloyl derivates 3, 6, 7, and 8 showed
moderate inhibitory effects against NO production with the
inhibition ratios (%) of 36.2, 35.1, 29.3 and 38.7 at 50 mM,
respectively, whereas 3-carboxy-indole derivates 1, 2, 4, and 5
showed potent inhibitory activities with IC50 values of 12.4, 8.3, 9.1,
0
0
NOE was observed between H-3 and H-4 , which suggested a trans
0
0
0
arrangement for H-3 and H-4 . The HMBC correlation from H-5
2H) to C-10 indicated that the 2-deoxy- -ribono- -lactone moiety
was connected to C-10. Thus, the structure of 1 was determined as
-O-3-carboxy-indole-2-deoxy- -ribono- -lactone.
Compound 2 was isolated as light brown gummy material. The
(
D
g
and 7.7 mM, respectively. This suggested that different sugars at C-
10 did not produce a significant effect on NO inhibitory activity. No
cytotoxicity was observed in compounds 1–8 treated cells (cell
viability >90%).
5
D
g
HRESIMS (negatve-ion mode) experiment revealed a pseudo-
ꢀ
molecular ion peak [MꢀH] at m/z 436.1248, in agreement with
3. Experimental
the molecular formula C20H23NO10. The spectroscopic properties of
2
were closely related to those of 1, except for the appearance of
3.1. General experimental procedures
0
signals due to Glc connected to C-3 of 2-deoxy-
in 2. This observation was supported by a relative downfield shift of
C-3 of 2 at
D-ribono-g-lactone
Optical rotations were measured on a JASCO P-1020 digital
polarimeter (Jasco, Tokyo, Japan). IR spectra were obtained on a
Bruker IFS-55 plus spectrometer (Bruker, Ettlingen, German). NMR
spectra were recorded on an Inova 500 spectrometer with TMS as
0
0
d
c 77.3 (C-3 of 1 at
d
c 69.6), and confirmed by HMBC
0
0
0
correlation between H-1 and C-3 . Thus, the structure of 2 was
determined as 5-O-3-carboxy-indole-3-O-(
-deoxy- -ribono- -lactone.
Compound 3 was isolated as colorless and amorphous powder.
b-D-glucopyranosyl)-
1
2
D
g
an internal standard, operating at 500 MHz for H and 125 MHz for
13
C (Bruker, Waltham, MA, USA). HR-ESI–MS data were obtained on
The HRESIMS (negatve-ion mode) experiment revealed a pseudo-
a Bruker-Daltonics APES-III 7.0 TESLA FTMS spectrometer (Bruker,
Billerica, MA, USA). GC was obtained on a SHIMADZU GC-14D.
Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co.,
Qingdao, China) were employed for thin layer chromatography.
Column chromatography was performed with silica gel (Merck,
Darmstadt, Germany) and C18 silica gel (150–200 mesh, Merck).
High performance liquid chromatography (HPLC) separation was
carried out on an octadecylsilanized column (YMC-pack ODS-A,
ꢀ
molecular ion peak [MꢀH] at m/z 501.1602, in agreement with the
1
molecular formula C22
H
30
O
13. The H NMR spectrum of 3 exhibited
the characteristic pattern of a isoferuloyl (=3-hydroxy-4-methox-
ycinnamoyl) moiety at 7.04 (d, J = 2.0 Hz, H-2), 6.94 (dd,
J = 8.0 Hz, H-5), 7.04(dd, J = 8.0, 2.0 Hz, H-6), 6.32 (d, J = 16.0 Hz, H-
d
H
7), 7.54 (d, J = 16.0 Hz, H-8), and 3.89 (s, 3H, OMe). Acid hydrolysis of
3
afforded isoferulic acid, glucose, and rhamnose, which were
detected by TLC comparison with authentic samples. The absolute
configuration of the monosaccharides was determined to be D for
250 ꢂ10 mm, i.d. 5
mm, YMC, Kyoto, Japan) with a photo-diode
array detector (Waters, Millford, MA, USA).

2
40
Q. Fu et al. / Phytochemistry Letters 17 (2016) 238–241
3
.2. Plant material
3.3.1. 5-O-3-carboxy-indole-2-deoxy-
D
-ribono-
ꢀ96, (c 0.5, MeOH); UV
g
-lactone (1)
2
5
White amorphous power, [
(MeOH):
2935, 1705, 1632, 1520, 1435; H and C NMR spectral data, see
a
]
D
ꢀ
1
The roots and rhizomes of Clematis mandshurica were collected
l
max 320, 284, 225, 210 nm; IR(KBr)
v
max (cm ): 3379,
1
13
in Jun 2015 in Chengdu, Sichuan Province, China. The identification
of the plant was performed by author (Qiang Fu). A voucher
specimen (CM 201506) is maintained in the herbarium of School of
Pharmacy and Bioengineering, Chengdu University.
ꢀ
Table 1. HRESIMS: m/z 274.0711 [Mꢀ H] (calcd for C14
5
H12NO ,
274.0715).
3
.3.2. 5-O-3-carboxy-indole-3-O-(
ribono- -lactone (2)
Light brown gummy material, [
MeOH): max 320, 284, 226, 211 nm; IR(KBr) vmax (cm ): 3378,
b
-
D
-glucopyranosyl)-2-deoxy-
D-
g
3.3. Extraction and isolation
a
]20
D
ꢀ92 (c 0.5, MeOH); UV
ꢀ1
(
2
l
The dried roots and rhizomes of C. mandshurica (10 kg) were
1
13
933, 1704, 1631, 1521, 1433; H and C NMR spectral data, see
extracted with EtOH. After removing the solvent, the residue
1661 g) was suspended in H O and extracted with n-BuOH (177 g).
The n-BuOH extract (177 g) was subjected to D101 porous polymer
resin column chromatography and eluted with H O and 30% and
0% EtOH, successively. The fraction that eluted with 30% EtOH
33.2 g) extract was subjected to silica gel column chromatography
CC) using eluent mixtures of CHCl
-MeOH (5:1 !1:2) to afford six
ꢀ
Table 1. HRESIMS: m/z 436.1248 [MꢀH] (calcd for C20
H22NO10,
(
2
4
36.1244).
2
3
.3.3. 9-O-
a
-
L
-rhamnopyranosyl-(1 !6)-
b
-
D
-glucopyranosyl
8
(
(
isoferuloyl ester (3)
Colorless amorphous power, [
a
]20
D
ꢀ28, (c 0.5, MeOH); UV
ꢀ1
3
(
MeOH): lmax 328, 299, 243 nm; IR(KBr) vmax (cm ): 3453, 1702,
fractions1-6 (3.9, 9.1, 7.8, 2.6, 4.5, and 3.9 g, respectively). Fraction 2
9.1 g) was subjected to by CC (Sephadex LH-20; MeOH) and prep-
HPLC (ACN-H
O, 20:80 ! 32: 68, 2.0 ml/min, UV detection at
30 nm) to furnish 1 (24 mg) and 3 (19 mg). Fraction 3 (7.8 g) was
subjected to 18 silica gel chromatography (MeOH–H O,
0:90 ! 70:30) to afford sub-fractions 1–5 (1.5, 3.2, 2.1, 0.6, and
.3 g, respectively). Sub-fraction 2 (3.2 g) was subjected to by CC
Sephadex LH-20; MeOH) and prep-HPLC (ACN-H O,
1
13
1654,1201; H and C NMR spectral data, see Table 1. HRESIMS: m/
(
ꢀ
z 501.1602 [MꢀH] (calcd for C22
29
H O13, 501.1608).
2
2
3
.4. Acid hydrolysis
C
2
1
0
Compound 2 and 3 (5 mg each) was heated in 0.5 ml of 2 M HCl
ꢃ
at 95 C for 10 h in a sealed tube. After filtration of the reaction
mixture, the filtrate was evaporated under vacuum. After addition
of H O, the acidic solution was evaporated again to remove HCl.
2
This procedure was repeated until a neutral solution was obtained,
which was finally evaporated and dried in vacuo to furnish a
monosaccharide residue. The sugar components obtained after
acid hydrolysis of 2 and 3 were analyzed by GC analysis of the
methyl sugar peracetates. The monosaccharide residue was
(
2
1
2:88 ! 18:82, 2.0 ml/min, UV detection at 230 nm), affording
compound 2 (26 mg), 4 (31 mg), and 5 (22 mg). Sub-fraction 3
(
2.1 g) was subjected to prep-HPLC (MeOH-H
UV detection at 230 nm), affording compound 6 (42 mg) and 7
19 mg). Sub-fraction 4 (0.6 g) was subjected to by CC (Sephadex
LH-20; MeOH) and prep-HPLC (ACN-H O, 30:70, 2.0 ml/min, UV
2
O, 34:66, 2.0 ml/min,
(
2
detection at 230 nm), affording compound 8 (21 mg).
dissolved in anhydrous pyridine (100 ml), 0.1 M L-cysteinemethyl
Table 1
1
H NMR and 13C NMR spectral data for compounds 1–3. (500 MHz for 1H NMR, 125 MHz for 13C NMR, in CD
OD).
3
No.
1
2
3
d
H
d
c
d
H
d
c
d
H
d
c
1
2
3
4
5
6
7
8
9
128.7
115.2
148.3
152.0
112.8
123.2
115.4
147.8
168.5
8.05 s
134.3
107.6
122.2
122.8
123.9
113.1
127.5
138.3
165.4
177.6
38.5
8.06 s
134.4
107.7
122.3
122.8
123.9
113.2
127.6
138.3
165.5
177.7
37.5
7.04 d (2.0)
8.10 m
7.21 m
7.22 m
7.47 m
8.12 m
7.22 m
7.23 m
7.48 m
6.94 d (8.0)
7.04 dd (8.0, 2.0)
6.32 d (16.0)
7.54 d (16.0)
1
0
0
0
1
2
5.58 d 8.0
3.45 dd (8.0, 9.0)
95.8
73.9
2.51 dd (3.5, 18.0)
2.75 dd (3.5, 18.0)
3.15 dd (8.0, 18.0)
4.57 m
4.71 m
4.33 m
3
.02 dd (7.0, 18.0)
0
0
0
3
4
5
4.44 m
4.58 m
4.22 dd (5.0, 12.0)
69.6
87.0
64.4
77.3
84.6
64.7
3.63 m
3.41 m
3.42 m
78.0
71.2
77.8
4
.42 dd (3.0, 12.0)
4.41 m
0
6
3.94 dd (2.0, 12.0)
67.8
3
.62 dd (6.0, 12.0)
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
4.41 d (8.0)
3.45 dd (8.0, 9.0)
3.34 m
3.35 m
3.29 m
103.9
74.8
78.0
71.3
77.8
62.4
4.72 br s
3.62 m
3.64 m
3.39 m
3.84 m
102.4
72.2
72.3
74.0
69.8
18.1
3.83 dd (2.0, 12.0)
1.21 d (6.0)
3
.58 dd (6.0, 12.0)
OMe
3.85 s 3H
56.5

Q. Fu et al. / Phytochemistry Letters 17 (2016) 238–241
241
ester hydrochloride (200 ml) was added, and the resultant was
Acknowledgement
ꢃ
warmed at 60 C for 2 h. The trimethylsilylation reagent HMDS–
TMCS (hexamethyldisilazane–trimethylchlorosilane–pyridine,
:1:10; Acros Organics, Geel, Belgium) was added and warmed
This work was supported by the National Natural Science fund
of China (Grant No. 81503234).
2
ꢃ
at 60 C for 30 min. To the mixture, the thiazolidine derivatives
were analyzed by GC for sugar identification. Separations were
carried out on a DB-5 column (30 m ꢂ 0.25 mm ꢂ 0.25 mm). Highly
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pure N
2
was employed as carrier gas (1.0 ml/min flow rate), and the
ꢃ
Flame Ionization Detector detector operated at 280 C (column
initial temperature was maintained at 100 C for 2 min and then
raised to 280 C at the rate of 10 C/min). The retention times of
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comparison with those of authentic standards.
ꢃ
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3
.5. Assay for inhibitory activity against LPS-induced NO production
Tested compounds were separately dissolved in DMSO, and
diluted with phosphate buffered saline (PBS) to a final concentra-
tion of 10 mM. Such solution was then diluted to various test
concentrations. RAW 264.7 macrophages were seeded in 24-well
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mandshurica. J. Nat. Prod. 69, 1591–1595.
plates (1 ꢂ10 cells/well), with cells co-incubated with the test
compounds and lipopolysaccharide (LPS, 1 mg/ml) for 24 h. The
amount of NO was assessed by determining the nitrite concentra-
tion in the cultured RAW 264.7 macrophage supernatants with
Shi, S.P., Tu, P.F., Dong, C.X., Jiang, D., 2006b. Alkaloids from Clematis manshurica
Rupr. J. Asian Nat. Prod. Res. 8, 73–78.
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Griess reagent. Aliquots of supernatants (100
in sequence, with 50 l 1% sulphanilamide and 50
naphthylethylenediamine in 2.5% H PO solution. Absorbances
at 570 nm were read using a microtiter plate reader.
m
l) were incubated,
m
m
l 0.1%
3
4