J Nat Med (2008) 62:236238

DOI 10.1007/s11418-007-0219-7

NOTE

New alkaloid from the aerial parts of Codonopsis clematidea

Shunsuke Ishida Mamoru Okasaka Freddy Ramos Yoshiki Kashiwada
Yoshihisa Takaishi Olimjon K. Kodzhimatov Ozodbek Ashurmetov

Received: 6 August 2007 / Accepted: 14 November 2007 / Published online: 11 January 2008
The Japanese Society of Pharmacognosy and Springer 2007

Abstract A new codonopsine-related alkaloid and 13

known compounds were isolated from the aerial parts of
Codonopsis clematidea (Campanulaceae). The structure of
the new compound was elucidated by two-dimensional
nuclear magnetic resonance and other spectral examinations.

which resulted in the isolation of a new codonopsinerelated alkaloid, designated as codonopsinol (1), together
with 13 known compounds. This paper describes the isolation and structural characterization of these compounds.

Keywords Codonopsis clematidea
Campanulaceae

Alkaloid
Codonopsine

Experimental
General experimental procedures

Introduction
Codonopsis spp., belonging to the Campanulaceae family,
are endemic to East Asia and are commonly called bonnet
bellflower. These plant species are perennial herbs, and
roots of the several species of this genus are used in Chinese traditional medicine as Codonopsis radix to treat
appetite loss, diarrhea, and vomiting. Chemical studies on
the genus Codonopsis so far examined contain alkaloids,
flavonoids, triterpenoids, saponins, and polyacetylenic
compounds [14]. The aerial parts, Codonopsis clematidea
(Schrenk) Clark are used in Uzbekistan to treat liver disease, hepatitis, and jaundice and are used in combination
with four other folk medicines to improve hepatic function
[5]. As our chemical studies on the medical plants in
Uzbekistan aimed at searching biologically active compounds, we examined the aerial parts of C. clematidea,
S. Ishida
M. Okasaka
F. Ramos
Y. Kashiwada

Y. Takaishi (&)
Graduate School of Pharmaceutical Sciences, University
of Tokushima, Shomachi 1-78, Tokushima 770-8505, Japan
e-mail: takaishi@ph.tokushima-u.ac.jp
O. K. Kodzhimatov
O. Ashurmetov
Institute of Botany and Botanical Garden, F. Khodzhaev,
St. 32, Tashkent 700143, Uzbekistan

123

Infrared (IR) spectra were recorded on a JASCO FT-IR420 spectrophotometer (PerkinElmer). Optical rotations
were measured with a DIP-370 digital polarimeter (JASCO). Nuclear magnetic resonance (NMR) [400 MHz for
1
H-NMR, 100 MHz for 13C-NMR, referenced to tetramethylsilane (TMS)] spectra were measured on a Bruker
Avance 400 Fourier transform spectrometer, and mass
spectrometry (MS) spectra were measured on an LCT
Premier 2695 (Waters). Column chromatographic supports:
silica gel 60N (63210 mm; Kanto Kagaku). Thin-layer
chromatography (TLC): silica gel 60F254 (Merck), Sephadex LH-20 (Amersham Pharmacia Biotech AB), Toyopearl
HW-40 (TOSOH), Amberlite XAD-2 (Organo). High-performance liquid chromatograohy (HPLC) columns: silica
gel (YMC-Pack SIL-06, 5 lm; YMC), gel-permeation
column (GPC) [Shodex; H2001 and H2002 (CHCl3), Asahi
pack GS-310 2G (MeOH)], and ODS [R-ODS-5 (YMC),
Mightysil RP-18 GP 250-20 (Kanto Kagaku).

Plant material
The aerial parts of C. clematidea were collected at Kashkadarya, Uzbekistan, in August 2002 (specimen number:
UTP: 040002).

J Nat Med (2008) 62:236238

Extraction and isolation

The air-dried aerial parts of C. clematidea (3.7 kg) were
extracted three times with MeOH at reflux. The MeOH
extract was concentrated under reduced pressure to give
a residue (261.0 g) that was partitioned successively
between n-hexane, EtOAc, n-BuOH, and H2O to yield
n-hexane-soluble fraction (91.6 g), EtOAc-soluble fraction (5.8 g), and n-BuOH-soluble fraction (25.0 g). The
n-BuOH-soluble fraction was applied on silica-gel column chromatography eluted with CHCl3MeOH (9:1) to
give ten fractions. Fraction 5 was subjected to Sephadex
LH-20 eluted with MeOH to afford a further nine fractions (frs. 5-15-9). Fraction 5-2 was purified by HPLC
(Mightysil-ODS column) [MeOHH2O (7:3)] and GPC
(MeOH) to give 1 (12.5 mg) and 2 (48.4 mg). Fraction 6
was repeatedly chromatographed over Amberlite XAD-2
chromatography [H2OMeOH (1:0 ? 0:1)], Sephadex
LH-20 (MeOH), HPLC (Mightysil-ODS column)
[MeOHH2O (7:3)], and GPC (MeOH) to yield 3
(25.1 mg) and 4 (7.5 mg). Fraction 5-3 was purified by
HPLC (Mightysil-ODS column) [MeOHH2O (7:3)] and
then silica gel [CHCl3:MeOH:H2O(7:3:0.5)] to give 5
(15.8 mg) and 6 (15.6 mg). The EtOAc-soluble fraction
was applied to silica-gel column chromatography eluting
with CHCl3MeOH (100:1 ? 10:1) to give seven fratctions (frs. 1117). Fraction 12 was repeatedly
chromatographed over Toyopearl HW-40 [CHCl3MeOH
(1:1)] and GPC (MeOH) and purified by PTLC to yield 8
(5 mg) and 14 (1 mg). Repeated chromatography of
fraction 16 with Sephadex LH-20 (MeOH), ODS
[MeOHH2O (65:35)], silica gel [CHCl3MeOH (10:1)],
and GPC (MeOH) furnished 9 (5 mg) and 13 (3 mg).
The hexane-soluble fraction was fractionated by silica-gel
column chromatography [n-hexane-EtOAc (9:1)] to give
ten fractions (frs. 1827). Repeated chromatography of
fraction 19 with silica gel [CHCl3acetone (9:1)],
Toyopearl HW-40 [CHCl3MeOH (1:1)], GPC (CHCl3),
and HPLC (CHCl3) gave 10 (19 mg) and 11 (20 mg).
Fractions 23 and 27 were purified separately by Topopearl HW-40 [CHCl3MeOH (1:1)], GPC (MeOH), and
HPLC (CHCl3) to furnish 12 (4 mg) and 7 (2 mg),
respectively.

Codonopsinol (1)
A white amorphous powder: [a]D-3.5 (c 0.2, MeOH). IR
(KBr) cm-1: 3419, 2933, 1594, 1519, 1261, 1141, 1024.
1
H-, and 13C-NMR spectral data (CD3OD) (Table 1).
HRESIMS m/z: 306.1319 [M + Na]+ (calcd. for
C14H21NO5Na, 306.1317).

237
Table 1 1H (dH, J in Hz) and
No.

13

C (dC) NMR dataa for 1 and 2

2
dC

dH

dH

dC

3.68 (d, 6.4)

76.8

4.26 (d, 5.2)

3.94 (dd, 6.4, 4.8)

85.5

4.33 (dd, 6.0, 4.0)

81.5

4.03 (t, 4.8)

79.9

4.01 (t-like)

83.0

3.11 (td, 8.4, 4.8)

72.3

3.67 (m)

76.9

69.1

135.1

7.03 (d, 2.4)

112.8

8
9

132.2
7.18 (d, 2.0)

113.8

150.1
150.7

151.1
151.9

10

6.89 (d, 7.6)

112.8

7.03 (d, 8.4)

113.0

11

6.91 (dd, 7.6, 2.4)

122.5

7.11 (dd, 8.4, 2.0)

124.0

12

3.84 (m)

60.7

1.50 (d, 7.2)

N-Me

2.22 (s)

35.1

2.57 (s)

35.4

OMe

3.83 (s)

56.5

3.87 (s)

56.6

OMe

3.81 (s)

56.4

3.85 (s)

56.5

11.8

13

Measured at 400 MHz ( H) and 100 MHz ( C) in CD3OD

Results and discussion

The MeOH extract of the aerial parts of C. clematidea was
partitioned successively with n-hexane, EtOAc, n-BuOH,
and H2O. The n-hexane-, EtOAc-, and n-BuOH-soluble
fractions were repeatedly subjected to silica gel, Sephadex
LH-20, Toyopearl HW-40C, and Amberlite XAD-2 chromatography with various solvent systems and to
preparative TLC and HPLC to yield compound 1 (12.5 mg)
and 13 known compounds (214). Compound 2 was
identified as (+)-codonopsine (2) [6], which has been isolated previously from this plant source. Compound 1, a
white amorphous powder, [a]D-3.5 (c 0.2, MeOH),
showed a hydroxyl band (3,419 cm-1) in the IR spectrum.
The 1H-NMR spectrum of 1 was closely correlated with
that of (+)-codonopsine (2) in showing signals due to a
1,3,4-trisubstituted aromatic ring [dH 6.89 (1H, d,
J = 7.6 Hz), 6.91 (1H, dd, J = 2.4, 7.6 Hz), 7.03 (1H, d,
J = 2.4 Hz)], two methoxyl groups [dH 3.81 and 3.83 (both
3H, s)] and a N-methyl group [dH 2.22 (3H, s)], together
with four methine protons at lower field. However, an
oxygen-bearing methylene signal [dH 3.84 (2H, m)]
appeared instead of the secondary methyl group observed
in 2. The 13C-NMR spectrum also resembled that of 2,
except for the observation of an oxygen-bearing methylene
carbon resonance (dC 60.7). The positive electron spray
ionization (ESI)-MS of 1 exhibited an [M + Na]+ ion peak
at m/z 306.1319, suggesting the molecular formula of
C14H21NO5, which was one oxygen more than that of 2.
These spectral observations suggested that 1 has a
hydroxymethyl group at C-5 position of (+)-codonopsine
(2). The 1H1H correlation spectroscopy (COSY)

123

238
Fig. 1 Structures of
codonopsinol (1), (+)codonopsine (2), and
radicamine A (15)

J Nat Med (2008) 62:236238

MeO
MeO

HO

7
6

9
10

MeO

11

OH
5

MeO
OH

12

examination revealed the presence of a CHCH(OH)

CH(OH)CHCH2O fragment structure. Furthermore,
heteronuclear multiple-bond correlations (HMBC) of H-3
(dH 3.94) to C-6 (dC 135.1); H-2 (dH 3.68) to C-7 (dC
112.8) and C-11 (dC 122.5); and N-methyl (dH 2.22) to C-2
(dC 76.8) and C-5 (dC 72.3) established the planer structure
of 1. Nuclear Overhauser enhancement (NOE) correlations
from H-3 to H-5, and H-2 to H-4 indicated that the relative
stereochemistry for the pyrrolidine moiety in 1 is the same
as that of (+)-codonopsine (2). The structurally related
pyrrolidine alkaloid radicamine A (15) was reported from
Lobelia chinensis (Campanulaceae) as an inhibitor of aglucosidase [7]. The absolute structure of 15 was recently
revised to be (2R,3R,4R,5R) by the enantioselective synthesis from D-xylose [8]. Comparison of the [a]D value
(+6.3) of N-methyllradicamine A with that (-3.5) of
codonopsinol suggested that the absolute stereochemistry
of codonopsinol was considered to be the same as (+)codonopsine (Fig. 1).
Compounds 314 were identified as foetidissimoside A
(3) [9], lobetyolin (4) [10], lobetyolinin (5) [11], (E)-2hexenyl-a-L-arabinopyranosyl-(1?6)-b-D-glucopyranoside
(6) [12], lariciresinol (7) [13], 1,6-hexanediol,3,4-bis(4hydroxy-3-methoxyphenyl) (8) [14], (6R,9S)-3-oxo-a-ionol-b-D-glucopyranoside (9) [15], taraxeryl acetate (10)
[16], 3b-acetoxyoleanan-12-one (11) [17], rubiprasin B
(12) [18], luteolin (13) [19], and apigenin (14) [20] by
comparison of their physical and spectral data with those
reported in the literature. This is the first report of the
isolation of compounds 314 from C. clematidea.

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