Phenolic alkaloids from the aerial parts of Dracocephalum heterophyllum

Article abstract of DOI:10.1016/j.phytochem.2012.06.021

Chemical examination of aerial parts of Dracocephalum heterophyllum resulted in isolation of phenolic alkaloids, including two flavonoidal alkaloids, drahebephins A and B, one imidazole alkaloid with a phenolic substituent, drahebenine, together with 15 other known compounds. Their structures were established by extensive spectroscopic data analyses. Due to the stereogenic center in the pyrrolidin-2-one ring, the flavonoidal alkaloids are chiral, although they were isolated as racemates. The enantiomers were separated by HPLC using a chiral phase and stereochemically characterized by circular dichroism (CD) spectroscopy. The structure of compound drahebenine was established by X-ray crystallography.

Full text of DOI:10.1016/j.phytochem.2012.06.021

Phytochemistry 82 (2012) 166–171
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenolic alkaloids from the aerial parts of Dracocephalum heterophyllum
⇑
Limei Wang, Shuqi Wang, Shuang Yang, Xiaojiang Guo, Hongxiang Lou, Dongmei Ren
Department of Natural Products Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 Wenhuaxi Road,
Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical examination of aerial parts of Dracocephalum heterophyllum resulted in isolation of phenolic
alkaloids, including two flavonoidal alkaloids, drahebephins A and B, one imidazole alkaloid with a phe-
nolic substituent, drahebenine, together with 15 other known compounds. Their structures were estab-
lished by extensive spectroscopic data analyses. Due to the stereogenic center in the pyrrolidin-2-one
ring, the flavonoidal alkaloids are chiral, although they were isolated as racemates. The enantiomers were
separated by HPLC using a chiral phase and stereochemically characterized by circular dichroism (CD)
spectroscopy. The structure of compound drahebenine was established by X-ray crystallography.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 January 2012
Received in revised form 2 May 2012
Available online 14 July 2012
Keywords:
Dracocephalum heterophyllum
Lamiaceae
Flavonoidal alkaloid
Imidazole alkaloid
Chiral HPLC
Absolute configuration
1. Introduction
pared and stereochemically characterized by circular dichroism
(CD) measurements. In addition, the structural investigation of
Plants of the genus Dracocephalum (Labiatae family) have been
documented as containing alkaloids, especially nitrogen-contain-
ing flavonoids which belong to a small class of natural products
(Ren et al., 2008). As a continuation of our research in discovery
of alkaloids from this genus, Dracocephalum heterophyllum Benth,
a perennial herb widely distributed in the western regions of China
was investigated (Wu and Li, 1977). Its aerial parts have long been
used in Tibetan medicine for the treatment of hypertension,
lymphadenitis, hepatitis and bronchitis. Some pharmacological
properties of the plant have been studied. In particular, it exhibited
an anti-hypoxic effect on rabbits fed on a diet supplemented with
the powdered herb under high-altitude exposure (Ma et al., 1994;
Xue et al., 1994; Zhao, 2002). Although there are some reports on
the constituents of D. heterophyllum, those studies mainly focused
on the essential oil composition (Singh et al., 2008; Zhang et al.,
2008). The current work led to the isolation of three novel phenolic
alkaloids (1–3) (Fig. 1) and 15 known compounds. Of these two
flavonoidal alkaloids, named drahebephins A and B (1–2), are chiral
compounds, although racemic, due to the presence of a stereogenic
center in the nitrogen-containing ring.
an imidazole alkaloid is presented.
2. Results and discussion
Drahebephins A (1) was isolated as a yellow amorphous
powder. Its molecular formula was determined as C19H15NO7 by
positive mode HRESIMS. The UV spectrum (kmax 255, 265 and
350 nm) suggested the presence of a flavonoid chromophore. The
IR data indicated the presence of phenolic hydroxy groups
(3300 cmꢀ1) and a carbonyl group (1650 cmꢀ1). The 1H NMR spec-
trum of 1 (Table 1) suggested the presence of a luteolin skeleton,
which was deduced from the typical ABX system observed for
the three B-ring protons at dH 6.87 (1H, d, J = 8.4 Hz), dH 7.39
(1H, d, J = 2.4 Hz) and dH 7.40 (1H, dd, J = 2.4, 8.4 Hz), together with
two singlets at dH 6.45 (1H, s) and dH 6.65 (1H, s), respectively. The
13C NMR spectrum (Table 1) displayed 19 signals, from which one
carbonyl resonance and 14 aromatic carbons for a luteolin moiety
were discerned. Based on the resonances at dH 7.53 (NH), 5.17 (dd,
J = 9.6, 4.8 Hz, H-500), 2.15 (m, 2H), and 2.35 (m, 2H) in the 1H NMR
spectrum and the four carbon resonances at dC 177.4, 31.1, 25.7,
and 47.0 in the 13C NMR spectrum, the presence of a pyrrolidone
moiety was determined. HMBC correlations from H-500 to C-6 (dC
112.6) and C-7 (dC 159.9) allowed the attachment of the 2-oxopyrr-
olidin-5-yl group at C-6 (Fig. 2). On the basis of these observations,
the structure of compound (1) was established to be 5,7,30,40-tetra-
hydroxy-6-(2-oxopyrrolidin-5-yl)-flavone.
Chromatography on a chiral phase has been shown to be an
effective tool for the separation of racemates (Bringmann et al.,
2001, 2008), by using this method, the analytical resolution of
the respective enantiomers was carried out. In order to establish
the absolute configurations the individual enantiomers were pre-
⇑
Although it contains a stereogenic center (C-500), compound 1
was found to be optically inactive, indicating that this compound
Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382019.
E-mail address: rendom@sdu.edu.cn (D. Ren).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.06.021

L. Wang et al. / Phytochemistry 82 (2012) 166–171
167
Fig. 1. Sructures of compounds 1–3.
configuration of the enantiomers, the CD spectra were measured,
providing almost mirror-image CD curves (Fig. 3). The faster elut-
ing enantiomer of 1 (peak A) had an obvious positive Cotton effect
(CE) at 204 nm, while the CD curve of the more slowly eluting one
(peak B) displayed a strong negative CE at 204 nm. In a previous re-
port of similar compounds, the arithmetically ‘‘isolated’’ CD curves
of C-500, showed a strong negative CE at 205 nm for the 500R-config-
uration and a positive CE for 500S-configuration (Ren et al., 2008).
Comparison with the experimental CD spectra of peaks A and B
gave good agreement between the 500S type and peak A, and be-
tween 500R type and peak B. From this pairwise match, the absolute
configurations of the two enantiomers were unambiguously
determined.
Table 1
1H and 13C NMR spectroscopic data for compounds 1–3.
Position 1a
2a
3b
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
122.6
111.4 7.41 (d, 1.8)
147.9
2
3
4
5
6
164.1
103.1 6.65 (s)
182.1
159.9
112.6
164.1
103.7 6.57 (s)
181.6
161.4
98.8 6.12 (s)
149.5
114.8 6.91 (d, 7.8)
121.8 7.34 (dd,
1.8, 7.8)
165.3
164.9
7
8
9
10
10
20
30
40
50
60
159.9
94.1 6.45 (s)
156.4
103.4
121.7
160.7
109.2
155.6
103.2
121.5
16.1 2.50 (s, 3H)
Drahebephins B (2) was also obtained as a yellow amorphous
powder. Its molecular formula was elucidated from HRESIMS data
113.6 7.40 (d, 2.4) 113.3 7.35 (d, 2.4)
98.8
146.3
150.5
145.7
149.6
22.9 1.50 (s, 3H)
22.9 1.50 (s, 3H)
116.5 6.87 (d, 8.4) 116.0 6.83 (d, 8.4)
119.4 7.40 (dd,
2.4, 8.4)
119.0 7.35 (dd,
2.4, 8.4)
200
300
177.4
177.2
31.1 2.15 (m,
2H)
31.8 2.09 (m,
2H)
400
500
25.7 2.35 (m,
2H)
25.6 2.29 (m,
2H)
47.0 5.17 (dd,
4.8, 9.6)
47.7 5.38 (dd,
4.8, 9.6)
NH
7.53 (s)
7.72 (s)
OCH3
55.0 3.93 (s, 3H)
a
DMSO-d6 was used as solvent.
CD3OD was used as solvent.
b
Fig. 2. Key HMBC (H ? C) correlations of 1.
is racemic as isolated. Nevertheless to investigate the existence of
possible enantiomers of drahebephins A (1) and the determination
of their ratio, a method for their enantiomeric resolution by HPLC
on a chiral phase was developed. Good results were achieved by
chromatography using a Chiralpak AS-H column. As expected,
two peaks were observed by UV detection. Integration of the sig-
nals gave a 1:1 enantiomeric ratio, confirming the racemic charac-
ter of 1 (Fig. 3).
The enantiomer separation on the chiral phase was successfully
extended to a semi-preparative scale, to give two enantiomeric
pure compounds. In order to attribute the respective absolute
Fig. 3. HPLC-UV analysis of 1 using a chiral phase (Chiralpak AS-H), and assignment
of the absolute configurations by CD spectroscopy.

168
L. Wang et al. / Phytochemistry 82 (2012) 166–171
Fig. 4. HPLC-UV analysis of 2 using a chiral phase (Chiralpak AS-H), and assignment of the absolute configurations by CD spectroscopy.
as C19H15NO7, the same as that of 1. Comparison of its 1H and 13C
NMR spectroscopic data with those obtained for 1 (Table 1) con-
firmed a strong structural similarity of these two compounds, the
only difference being the substitution of the pyrrolidone ring at
C-8 in 2 instead of C-6, this was further confirmed by analysis of
H3C
N
CH3
CH3
N
HO
the HMBC data. Therefore, structure
2 was established as
OCH3
5,7,30,40-tetrahydroxy-8-(2-oxopyrrolidin-5-yl)-flavone.
For a complete stereochemical attribution of drahebephins B,
the above-mentioned method for resolution of the enantiomers
of 1 was applied to compound 2. Observation of two LC–UV peaks
with a 1:1 intensity ratio determined the racemic nature of 2. The
two enantiomers were obtained by chiral HPLC as described above.
CD measurements gave mirror-image CD curves and allowed the
determination of the absolute configuration of the two enantio-
mers (Fig. 4). Comparison of the CD spectrum of the faster eluting
peak (peak A) of 2 with the corresponding one of 1 established a
high similarity, suggesting that faster eluting peaks to corre-
sponded to the 500S-configured enantiomer. Conversely, the CD
spectrum of peak B (more slowly eluting peak) agreed well with
that for the 500R configuration, thus permitting assignment of the
absolute configurations to the respective enantiomers.
Fig. 5. Key HMBC (H ? C) correlations of 3.
Drahebenine (3) was obtained as colorless crystals with a
molecular formula of C13H16N2O2 by HRESIMS ([M+H]+), indicating
7 degrees of unsaturation. The 1H spectrum of 3 (Table 1) showed a
three-proton ABX system (dH 6.91, 1H, d, J = 7.8 Hz; 7.34, 1H, dd,
J = 1.8, 7.8 Hz; and 7.41, 1H, d, J = 1.8 Hz) characteristic of a trisub-
stituted aromatic ring; singlets at 3.93 (3H, s), 2.50(3H, s) and 1.50
(6H, s) typical of one methoxy and three methyls, respectively. The
13C NMR (Table 1) and HMQC spectra of 3 indicated the presence of
six aromatic carbons due to the trisubstituted aromatic ring, two
additional sp2 carbons, three methyl carbons, one methoxyl carbon
and one quaternary carbon. The observed HMBC correlations of dH
3.93 (OCH3-3) to dC 147.9 (C-3) and dC 149.5 (C-4), of dH 7.41 (H-2)
to dC 147.9 (C-3) and 122.6 (C-1), and of dH 6.91 (H-5) and 7.34 (H-
6) to dC 149.5 (C-3) showed 3-methoxy-4-hydroxybenzyl moiety.
The two deshielded sp2 carbons resonating at dC 165.4 (C-7) and
164.9 (C-8), one sp3 quaternary carbon at dC 98.8 (C-20) were attrib-
uted to tetrasubstituted 2H-imidazole ring. In the HMBC spectrum,
cross-peaks from H-2 and H-6 to C-7 established the connection of
imidazole moiety with the benzene ring at C-7. HMBC correlations
between the methyl resonances at dH 2.50 (H3-9) and 1.50 (H3-40,
H3-50) assigned the substituted positions of the methyls (Fig. 5).
A single crystal of 3 was obtained and subjected to X-ray crystallo-
graphic diffraction analysis (Fig. 6). The result unambiguously
Fig. 6. X-ray structure of 3.
confirmed the structure of 3. Therefore, compound 3 was deter-
mined as a conjugate of methoxyphenol and trimethyl-2H-imidaz-
ole, i.e. 2-methoxy-4-(2,2,5-trimethyl-2H-imidazol-4-yl)phenol,
which was named drahebenine.
In addition, 15 known compounds were also isolated: ursolic
acid (4) (Sang et al., 2002), oleanolic acid (5) (Mimaki et al.,
2004), betulinic acid (6) (Cichewicz and Kouzi, 2004), betulin (7)
(Gherraf et al., 2010), 2
1989), diosmetin (9) (Park et al., 2007), luteolin (10) (Flamini
et al., 2001), luteolin-5-O-b- -glucoside (11) (Veit et al., 1990),
a-dihydroxy ursolic acid (8) (Kuang et al.,
D
luteolin-7-O-rutinoside (12) (Kim et al., 2000), apigenin-7-O-ruti-
noside (13) (Baris et al., 2011), rosmarinic acid (14) (Dapkevicius
et al., 2002), methyl rosmarinate (15), ethyl rosmarinate (16)
(Woo and Piao, 2004), 3-methoxy-4-hydroxybenzoic acid (17),
and 2-hydroxybenzoic acid (18). Their structures were identified
by comparison of NMR and MS spectroscopic data with those pre-
viously reported in the literatures.

L. Wang et al. / Phytochemistry 82 (2012) 166–171
169
It is reported that luteolin, the parent compound of 1 and 2, pos-
M at 48 h for some cancer
and chiral HPLC were carried out with Agilent HP 1100 instrument,
using Agilent Eclipse XDB-C18 (5
sesses anticancer activity with IC50 ꢁ40
l
l
M, 9.4 ꢂ 250 mm) and Chiralpak
cell lines (Attoub et al., 2011; Cai et al., 2011), as well as the ability
to sensitize cancer cells to chemotherapeutic agents (Amin et al.,
2010). Thus the cytotoxic activity of drahebephin A on A549 lung
cancer cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay was tested. However, it did not show
AS-H chiral column (Daicel) respectively. Silica gel (200–300 mesh)
and Sephadex LH-20 gel (Amersham Biosciences) were used for
column chromatography (CC). All solvents used for extractions
and chromatographic separations were of analytical grade, with
the exception of HPLC grade solvents used for HPLC separations.
any cytotoxicity at concentrations from 10 to 100 lM, while lute-
olin showed almost the same effect as reported in the same assay.
In a search for an increasing chemotherapy effect, 1 did not show
any synergestic effect when combined with cisplatin, however
luteolin increased the effect of cisplatin at a concentration of
10 lM, similar to results previously reported in the literature (Tang
et al., 2011). It seemed that substitution with the pyrrolidone ring
4.2. Plant material
The aerial parts of D. heterophyllum were collected in Qinghai
Province, China, in July 2008. Identification was carried out
by Professor Yarong Liu of Qinghai Institute for Drugs Control,
China. A voucher specimen (Accession No. DH-2008-001) has
been deposited in School of Pharmaceutical Sciences, Shandong
University.
decreased the anticancer activity of luteolin.
3. Conclusions
4.3. Extraction and isolation
To date, more than 240 compounds have been isolated and
identified from the genus Dracocephalum, with triterpenoids and
flavonoids being the typical constituents (Zeng et al., 2010). As a
result of present study, D. heterophyllum has been shown to be
possess a chemical profile similar to those of other species from
Dracocephalum, i.e. triterpenoids and flavonoids represented as
major components. Current phytochemical investigations also led
to isolation of two novel flavonoidal alkaloids and one imidazole
alkaloid from D. heterophyllum. Flavonoidal alkaloids have a lim-
ited distribution in nature with only less than 30 compounds re-
ported until now. The newly-found drahebephins A (1) and B (2)
further enlarge the spectrum of this class of natural compounds.
Isolation of alkaloids has previously been achieved from only
two Dracocephalum species, D. rupestre and D. tanguticum, the pres-
ent study represents the third example from the same genus. The
alkaloids from D. tanguticum have been elucidated as spermidine
glycosides (Wang et al., 2009). However, both D. rupestre and D.
heterophyllum produce structurally similar flavonoidal alkaloids.
The main difference is that D. rupestre accumulates conjugates of
flavanones and pyrrolidone, whereas D. heterophyllum contains
conjugates of flavones and pyrrolidone. More discovery of flavonoi-
dal alkaloids from the genus Dracocephalum may contribute to
enhancing our understanding of the chemotaxonomy and evolu-
tion of the genus.
Air-dried aerial parts of D. heterophyllum (5.0 kg) were extracted
with EtOH–H2O (95:5, v/v, 20 L 3ꢂ) at room temperature. After re-
moval of solvents under reduced pressure, the crude extract
(450 g) was suspended in H2O and partitioned successively with
petroleum ether, CHCl3 and n-BuOH. The CHCl3 fraction was evap-
orated in vacuo to give a residue (69.2 g), which was subjected to
silica gel CC, eluting with solvent of increasing polarity (CHCl3–
MeOH, 1:0 ? 0:1) to give five major fractions (CH1–CH5). Fraction
CH2 was subjected to silica gel CC using a gradient system of
CHCl3–MeOH (1:0 ? 1:1) to produce five subfractions (CH2A–
CH2E). Subfraction CH2A was purified by silica gel CC using a
step-wise gradient of CHCl3–MeOH (20:1, 10:1, 9:1 5:1 and 1:1)
to afford compounds 4 (3.0 g), 5 (3.5 g), 6 (10.0 mg) and 7
(8.0 mg). Fraction CH3 was purified by Sephadex LH-20 (CHCl3–
MeOH, 1:1) CC to yield compound 8 (200 mg). Fraction CH5 was
separated on a column of Sephadex LH-20 (CHCl3–MeOH, 1:1),
and then purified by semi-preparative HPLC, with MeOH–H2O
(65:35, v/v) as mobile phase, to produce compound 3 (11 mg).
After concentration, an aliquot of the n-BuOH-soluble fraction
(80 g) was separated by silica gel CC, eluted with a gradient of
CHCl3–MeOH (1:0 ? 0:1) to give nine major fractions (BU1–BU9).
Fraction BU2 (3.0 g) was further separated by a silica gel CC, eluted
with CHCl3–MeOH (20:1 ? 1:1) in a gradient to afford eight sub-
fractions (BU2A–BU2H). Subfraction BU2C was then purified by
Sephadex LH-20 (CHCl3–MeOH, 1:1) CC, followed by semi-prepara-
tive HPLC (MeOH–H2O, 63:37) on a C18 column to afford 1 (7 mg)
and 2 (4 mg). Separation of the four single stereoisomers of 1
and 2 was achieved by HPLC on a Chiralpak AS-H column. The sol-
vent system used for the isolation of the two enantiomers of 1 con-
sisted of CH3CN–iPrOH (92:8), while the mobile phase for the
separation of the two enantiomers of 2 consisted of CH3CN–iPrOH
(98:2). The flow rate was 0.7 mL/min at room temperature and
detection was carried out at 210 nm. Fraction BU3 was separated
on a silica gel column using a step-wise gradient of CHCl3–MeOH
(10:1, 8:1, 5:1 and 1:1) to afford four subfractions (BU3A–BU3D).
Subfraction BU3A was further purified by silica gel (CHCl3–MeOH,
9:1) CC to yield compounds 17 (11.0 mg) and 18 (5.0 mg). Subfrac-
tion BU3C was then further purified by Sephadex LH-20 (CHCl3–
Enantiomeric analysis of compounds 1 and 2 has been achieved
by chromatography on a chiral AS-H HPLC phase, thereby indicat-
ing that the two compounds are racemic. All the four enantiomers
were obtained by chiral HPLC and the absolute configuration at C-
500 of the pyrrolidone ring were unambiguously assigned by CD
measurements. Experimental CD contributions of the stereogenic
carbon of the pyrrolidin-2-one ring are reported for the first time
in the field of nitrogen containing flavonoids. The structure of dra-
hebenine (3) was confirmed by X-ray crystallographic analysis.
4. Experimental
4.1. General
UV spectra were recorded on a Shimadzu UV-2550 UV–Visible
spectrophotometer. IR spectra were recorded on a Nicolet NEXUS
470 FT-IR spectrometer with KBr disks. NMR spectra were mea-
sured on a Brucker AV-600 spectrometer in CD3OD or DMSO-d6
with TMS as internal standard. HRESIMS were carried out on a
LTQ-Orbitrap XL instrument. CD spectra were measured on a
Chirascan spectropolarimeter, using a 5 mm cell. Melting points
(mp) were determined with an X-4 micro-melting point apparatus
(Beijing Tech Co., Ltd.) and are uncorrected. Semi-preparative HPLC
MeOH, 1:1) CC to afford compounds 9 (8.0 mg) and 10
(120.0 mg). Fraction BU4 was applied to a silica gel column, eluted
with CHCl3–MeOH (4:1) to afford three subfractions (BU4A–BU4C).
Subfraction BU4B was purified by Sephadex LH-20 (EtOH) CC to
give compound 13 (8.0 mg). Fraction BU6 was fractionated by
Sephadex LH-20 (EtOH) CC to afford two subfractions (BU6A and
BU6B). Subfraction BU6A was then purified by semi-preparative
HPLC (MeOH–H2O, 50:50) on a C18 column to yield compounds
11 (7.0 mg) and 12 (85.0 mg). Fraction BU7 was separated by silica

170
L. Wang et al. / Phytochemistry 82 (2012) 166–171
gel (CHCl3–MeOH, 4:1) CC to yield three subfractions (BU7A–
BU7C). Subfraction BU7A was further separated by semi-prepara-
tive HPLC (MeOH–H2O, 60:40) on a C18 column to yield compounds
14 (12.0 mg), 15 (15.0 mg) and 16 (10.0 mg).
Acknowledgments
Financial support of the National Natural Science Foundation
(Nos. 30973622 and 81173528) and Shandong Province Natural
Science Foundation (No. Y2007C038) are gratefully acknowledged.
4.4. Drahebephins A (1)
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4.6. Drahebenine (3)
Colorless crystals; mp 179–180 °C; UV (MeOH) kmax: 270,
348 nm. IR (KBr) mmax cmꢀ1: 3093, 1506, 1427, 1267, 1218, 1190,
1030, 912, 872, 778 cmꢀ1. For 1H and 13C NMR spectroscopic data,
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4.7. Cytotoxicity assay
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8 ꢂ 103 cells per well were seeded in a 96-well plate and incu-
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of compounds for 48 h followed by the addition of 20 ll of 2 mg/
mL MTT into the medium. After incubation at 37 °C for 4 h, the
plate was centrifuged and the medium was removed. For each well,
DMSO (100 ll) was added and crystals were dissolved by shaking
the plate at room temperature. Absorbance was measured by a
plate reader at 570 nm. Triplicate wells were used for each sample
and the experiments were repeated at least three times to get
means and standard deviations. Cisplatin was used as positive
control.
4.8. X-ray crystallography of compound 3
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was used for analysis. Single crystal X-ray analysis established
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summarized as follows: C13H16N2O2, formula wt 232.28, ortho-
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c = 9.1690 (18) Å, V = 1181.6 (4) Å3, Z = 4, Dc = 1.306 Mg/m3.
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