Isoquinoline alkaloids isolated from Corydalis yanhusuo and their binding affinities at the dopamine D1 receptor

Article abstract of DOI:10.3390/molecules13092303

Bioactivity-guided fractionation of Corydalis yanhusuo has resulted in the isolation of eight known isoquinoline alkaloids - tetrahydropalmatine, isocorypalmine, stylopine, corydaline, columbamine, coptisin, 13-methylpalmatine, and dehydrocorybulbine. The

Full text of DOI:10.3390/molecules13092303

Molecules 2008, 13, 2303-2312; DOI: 10.3390/molecules13092303
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molecules
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Communication
Isoquinoline Alkaloids Isolated from Corydalis yanhusuo and
Their Binding Affinities at the Dopamine D1 Receptor
Zhong-Ze Ma 1, Wei Xu 2, Niels H. Jensen 3,4, Bryan L. Roth 3,4 Lee-Yuan Liu-Chen 2 and
David Y. W. Lee 1,*
1
Bio-Organic and Natural Products Laboratory, McLean Hospital, Harvard Medical School, 115
Mill Street, Belmont, MA 02478, USA
2
Department of Pharmacology and Center for Substance Abuse Research, School of Medicine,
Temple University, 3420 N. Broad St, Philadelphia, PA 19140, USA;
E-mail: wxu00001@temple.edu (W. X.), lliuche@temple.edu (L-Y. L-C.)
3
Department of Pharmacology, University of North Carolina Medical School, Chapel Hill, NC
27599, USA; E-mail: niels_h_jensen@hotmail.com (N-H. J.)
4
Center for Neurobiology Division of Medicinal Chemistry and Natural Products, and NIMH
Psychoactive Drug Screening Program, University of North Carolina Medical School, Chapel Hill,
NC 27599, USA; E-mail: bryan_roth@med.unc.edu (B-L. R.)
* Author to whom correspondence should be addressed; E-mail: dlee@mclean.harvard.edu;
Tel.: +1-617-855-2038; Fax: +1-617-855-2040.
Received: 29 August 2008; in revised form: 15 September 2008 / Accepted: 16 September 2008 /
Published: 25 September 2008
Abstract: Bioactivity-guided fractionation of Corydalis yanhusuo has resulted in the
isolation of eight known isoquinoline alkaloids - tetrahydropalmatine, isocorypalmine,
stylopine, corydaline, columbamine, coptisin, 13-methylpalmatine, and dehydro-
corybulbine. The tertiary alkaloids were further analyzed by chiral HPLC to determine the
ratios of d-and l-isomers. The isolated compounds were screened for their binding
affinities at the dopamine D1 receptor. Isocorypalmine had the highest affinity (Ki = 83
nM). The structure-affinity relationships of these alkaloids are discussed.
Keywords: Corydalis yanhusuo; Isoquinoline alkaloids; d/l Ratio; Chiral HPLC;
Dopamine receptor

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Introduction
Alcohol and drug abuse are major social and medical problems that impose a significant burden on
society [1-2]. Because of the complexity of drug dependence and the lack of effective remedy,
especially for relapse, the development of pharmacotherapy for its treatment has been a serious
challenge [3]. Despite a great deal of effort in developing effective therapies, only few medications
have been approved by the U.S. Food and Drug Administration [2, 4]. These medications have shown
only limited efficacy, serious side effects and poor compliance in patients [5-8], therefore, an
alternative approach, the systematic evaluation of traditional herbal medicines, may provide new drug
candidates for the treatment of alcohol and drug abuse. Chinese herbal medicines have been used
historically in the treatment of alcohol and drug abuse [4, 9]. Many are still prescribed in China and
Southeast Asia for this purpose, and their clinical efficacy of some medications has recently been
documented [10]. NPI-025 [11] is a herbal formula used clinically to treat opioid addiction in Hong
Kong. It is composed of five Chinese herbs, including Rhizoma Corydalis (Yan Hu Suo), Rhizoma Et
Radix Notopterygh (Qiang Huo), Ramulus Uncariae Cum Uncis (Gou Teng), Rhizoma Chuanxiong
(Chuan Xiong) and Radix Aconiti Lateralis Preparata (Fu Zi, treated with hot strong base to reduce
toxicity) [12-13].
The dried tuber of Corydalis yanhusuo (Papaveraceae) is one of key ingredients in NPI-025. C.
yanhusuo has been used traditionally to promote blood circulation, reinforce vital energy, and alleviate
pain such as headache, chest pain, epigastric pain, abdominal pain, backache, arthralgia,
dysmenorrheal pain, or trauma, and is officially listed in the Chinese Pharmacopoeia [14-16]. Previous
phytochemical and pharmacological studies have identified several alkaloids as the active secondary
metabolites of the plant [16]. dl-Tetrahydropalmatine (dl-THP), one of the major active alkaloids, has
been found to be a neuroactive alkaloid [17-19]. It has been listed in the Chinese Pharmacopoeia since
1977 as an analgesic with sedative and hypnotic effects. Recent studies have demonstrated that l-THP
inhibits opiate tolerance and withdrawal syndromes in rats [20-21]. It was also reported that l-THP
significantly inhibits cocaine- or methamphetamine-induced conditioned place preference [22-23]. In
addition, l-THP inhibited cocaine-triggered reinstatement [24] and the rewarding effects of cocaine in
rats as measured by cocaine self-administration [24-25] and intracranial self-stimulation [25]. In a
recent double-blind clinical trial in China, Yang et al. [10] found that treatment of 119 heroin-only
dependent in-patients with l-THP (60 mg orally twice a day) for one month significantly reduced
heroin craving and withdrawal symptoms. These findings suggest that l-THP could be an excellent
drug candidate for treatment of drug addition.
As part of our ongoing investigation of alternative therapies for substance addiction [11, 26], we
initiated a comprehensive fractionation study guided by dopamine receptor binding assays in an
attempt to find new dopamine receptor agonists or antagonists from C. yanhusuo. Herein we report the
isolation of eight isoquinoline alkaloids, their D1 receptor binding activities and their stereochemistry
by chiral HPLC method.

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Results and Discussion
The 70% aqueous acetone extract of C. yanhusuo were subjected to sequential extraction with
hexane, ethyl acetate, butanol, methanol and water. In our initial biological study as shown in Table 1,
the hexane, ethyl acetate, butanol and methanol extracts at 50 mg/mL showed moderate binding
activity at rat dopamine D1 receptor (rD1R) stably expressed in CHO cells, compared with the positive
control SKF82958 [27] (10 μM), while the water extract had no activity.
Table 1. Displacement of [3H]SCH 23390 binding to membranes of CHO cells stably
expressing the rD1R.
Sample I.D.
hexane extract
AcOEt extract
BuOH extract
MeOH extract
water extract
SKF82958
Concentration
50 mg/mL
50 mg/mL
50 mg/mL
50 mg/mL
50 mg/mL
10 μM
Displaced [3H]SCH 23390
100%
96%
97%
88%
-2%
99%
The hexane, ethyl acetate, butanol and methanol extracts were chromatographed on silica gel
columns to give eight known isoquinoline alkaloids (Figure 1), tetrahydropalmatine (1) [28],
isocorypalmine (2) [28], stylopine (3) [29], corydaline (4) [30], columbamine (5) [31], coptisin (6)
[31], 13-methylpalmatine (7) [31] and dehydrocorybulbine (8) [32]. The tertiary alkaloids 1, 2 and 3
have a chiral center at the C-14 position, and may consist of two optical isomers. Compound 4 has two
1
chiral centers at the C-13 and C-14 positions, and the H-NMR spectrum of 4 displayed the H-14
methine as a doublet at δ 3.68 (J = 2.7 Hz), indicating the protons at H-13 and H-14 are cis oriented
[30]. Following the reported method [17-18], compounds 1, 2, 3 and 4 were analyzed by chiral HPLC.
As shown in Table 2, the ratios of d- and l-isomers of 1, 3 and 4 are 3:1, 1:10 and 10:1, respectively,
while 2 gave only one peak in its chiral HPLC (Figure 2). In order to further confirm the
stereochemistry of 2, l-2 was synthesized by demethylation of l-THP (l-1) with BBr3 in CH2Cl2
(Scheme 1). The reaction time is critical for a good yield. After one hour, the main product was l-2,
while after 2 h the main product was found to be the l-scoulerine (9). Compounds l-2 and 9 were
difficult to be separated by silica gel column chromatography. However, based on its solubility, l-2 can
be readily purified by recrystalization from methanol. Following the same synthetic procedure, dl-2
was also prepared from dl-THP (dl-1). Comparison of the chiral HPLC chromatogram of 2 with those
of l-2 and dl-2 (Figure 2) confirmed that compound 2 was the l-enantiomer.
Scheme 1. Synthesis of l-2 from l-1.

Molecules 2008, 13
Figure 1. Isoquinoline alkaloids isolated from C. yanhusuo.
2306
Figure 2. Chromatograms obtained from chiral separation of 2 (A), synthetic l-2 (B) and dl-2 (C).
HPLC conditions: Waters 1525 Binary HPLC Pump; Waters 2487 Dual λ Absorbance
Detector; Chiralcel OD Column (4.6 x 250 mm); 50% EtOH as mobile phase; Wavelength,
230 nm; Sample concentration, 0.5 mg/mL; Injection volume, 5 µL
The isolated alkaloids were tested for dopamine receptor binding as reported previously [11].
Compounds 1, 2, 3, 5, 7 and 8 inhibited [3H]SCH 23390 binding to human D1 receptor (hD1R) by more
than 50% at 10 μM, and their binding affinities were determined (Table 2). The l-enantiomer of 1 (l-

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THP) has a D1 affinity of 94 nM while the 3:1 mixture of the d- and l-enantiomers 1 has a much
weaker affinity of 1.3 μM. It has been reported that l-1 has analgesic activity with a more potent
tranquilizing effect than the d isomer [19, 33]. Previous studies have demonstrated that l-1 acts on
dopamine receptors, but d-THP does not [34, 35]. Compound l-1 is a non-subtype selective antagonist
at dopamine receptors[35-37].
Of the isolated alkaloids, compound 2 has the highest D1 affinity with a Ki of 83 nM, comparable in
potency to l-1. Jin et al. [35, 37] reported that l-THP derivatives with one hydroxyl group at C-2 and
two hydroxyl groups at C-2 and C-9 or C-2 and C-10 were more potent at dopamine receptors than l-1.
On the other hand, Guo et al. [38] reported that l-1 was readily metabolized to desmethylated l-THPs
in rats.
Table 2. Binding affinities of the purified alkaloids for the [3H] SCH 23390 labeled the
hD1R in transiently transfected HEK-T cells.
Compound
ratio of d:l-isomers
Ki (nM)a
1,293 ± 71
83 ± 5
1,043 ± 66
>10,000
1
2
3
4
5
6
3:1
0:1
1:10
10:1
-
4,112 ± 274
>10,000
-
7
8
-
-
0:1
3,867 ± 235
1,836 ± 200
94 ± 9
l-THP
a Each value represents the mean ± SEM of three independent experiments performed in duplicate
As shown in Table 2, the quaternary alkaloids (5 and 6) exhibited weaker binding affinities than the
corresponding tertiary alkaloids (2 and 3). Comparison of the binding affinities of 7 and 8 with that of
5 revealed that the methyl group at C-13 did not affect the overall binding activity at the D1 receptor.
In order to obtain enough pure alkaloids for biological and pharmacological studies, another larger
batch of crude extract of C. yanhusuo was fractionated. Compounds 1, 2, 3 and 4 were isolated from
the new extract. Subsequent chiral HPLC analysis showed that the ratios of d- and l-enantiomers in
newly isolated 2 and 3 remained the same as those of previous isolated 2 and 3, while the ratios in both
1 and 4 were changed to 1:1. The changes of these components in different extracts of C. yanhusuo
indicated that different geographical regions, seasons of harvest, storage, or extraction conditions
could affect the chemical composition significantly. As evidenced in our case, quality control of the
active optical isomers will be very important for future biological, pharmacological, and clinical
studies of C. yanhusuo.
Conclusions
The mesolimbic dopaminergic system has been shown to play an important role in drug abuse [39].
Compounds targeted to these dopamine receptors can provide a rational treatment of drug abuse [40].
The stereochemistry of l-isocorypalmine (2) and the d/l ratio of 1, 3, and 4, were established
unambiguously by using a chiral HPLC method along with concise chemical synthesis. In addition,

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this convenient and effective synthesis for selective cleavage the methoxy group at C-2 position
allows the preparation of l-isocorypalmine in a large amount from readily available l-
tetrahydropalmatine for animal study. The present study demonstrates that isoquinoline alkaloids are
the principal constituents for the binding activities of C. yanhusuo extracts at the dopamine D1
receptor. l-Isocorypalmine (2) showed the highest affinity and is a promising drug candidate. This
study provides an important pharmacological basis to support the traditional use of C. yanhusuo in the
treatment of heroin addiction in China. Its complete pharmacological characterization is under
investigation and will be reported in due course.
Experimental
General
The NMR spectra were recorded on a Varian VXR300 spectrometer with TMS as the internal
standard. EI-MS spectra were obtained on a HP5972 Series Mass spectrometer. HPLC was performed
on a Waters (Milford, MA, USA) system comprised of a 1525 Binary HPLC Pump equipped with a
Waters 2487 Dual λ Absorbance Detector set at a wavelength of 230 nm, a model 7725i sample
injector equipped with a 5 μL loop and a Waters Breeze software package for data collection.
Compounds were separated on a Chiralcel OD Column (4.6 x 250 mm). The mobile phase was 50:50
(v/v) ethanol–water. All analysis was performed at a flow-rate of 0.5 mL/min with detection at 230
nm. The mobile phase was filtered through a 0.45 μm filter and degassed. Separations were performed
at room temperature. Silica gel (Fisher Scientific, USA) were used for column chromatography. TLC
was performed on precoated silica gel 60 F254 plates (Merck, Germany).
Plant material
The dried tubers of C. yanhusuo were collected from Qianxian Town, Dongyang District, Zhejiang
province, P.R. China in May, 2005. Its botanical identification was confirmed by Dr. Shilin Chen, the
Institute of Medicinal Plant Development (IMPLAD), Beijing, P.R. China. A voucher specimen has
been deposited in the IMPLAD, Beijing, P.R. China.
Extraction and isolation
The dried tubers of C. yanhusuo (30 kg) were powdered and extracted with 70% aqueous acetone
for three times at room temperature, and the solution was evaporated under reduced pressure to give a
residue (2.2 kg). The 70% aqueous acetone extract (200 g) was subjected to sequential extraction with
hexane, ethyl acetate, butanol, methanol and water. The combined part (3 g) of the above hexane, ethyl
acetate and butanol extracts was chromatographed over silica gel (100 g) using CH2Cl2 with increasing
amounts of MeOH (10:1, 5:1, 3:1, 1:1 and 1:2) to give five fractions A-E. Fraction A was further
purified by silica gel column, with a gradient of hexane and acetone to afford 1 (20 mg), 2 (15 mg), 3
(10 mg) and 4 (15 mg). Fraction B was chromatographed over silica gel column with a gradient of
CH2Cl2 and MeOH to afford 5 (10 mg), 7 (250 mg) and 8 (3 mg). Fraction C was purified by silica gel

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column, with a gradient of CH2Cl2 and MeOH, to afford 6 (8 mg). In a similar manner, the MeOH
extract (8 g) was separated by silica gel column chromatography using CH2Cl2 with increasing
amounts of MeOH to give 5 (20 mg) and 7 (500 mg).
Synthesis of l-isocorypalmine (2)
To a stirred CH2Cl2 solution (10 mL) of l-tetrahydropalmatine (l-1, 355 mg, 1.0 mmol), BBr3
solution (1 mM in CH2Cl2, 5 mL) was added. The reaction was stirred at -78 ºC for 1 h, and then was
warmed to room temperature. The mixture was diluted with CH2Cl2 (50 mL) and washed with excess
saturated NaHCO3. The organic extract was dried (MgSO4), filtered and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (CH2Cl2 and MeOH, 20:1),
followed by recrystallization in MeOH, to give l-2 (150 mg) and l-scoulerine (9, 40 mg).
Competitive inhibition of [3H] SCH23390 binding to dopamineD1 receptor
Chinese hamster ovary cells (CHO) stably transfected with HA-tagged rat D1 dopamine receptor
(rD1R) were grown in 100-mm culture dishes in Dulbecco's modified Eagle's medium F12 HAM
supplemented with 10% fetal calf serum, 0.1 mg/mL hygromycin B, 100 units/mL penicillin and 100
µg/mL streptomycin in a humidified atmosphere consisting of 5% CO2 and 95% air at 37°C. Binding
to membranes prepared from CHO cells stably transfected with the rD1R was conducted with
[3H]SCH23390 (0.2 nM) in 50mMTris-HCl buffer containing 1 mM EGTA (pH 7.4) (TE buffer) at
room temperature for 1 h in duplicate. Nonspecific binding was defined as binding in the presence of
Fluphenazine (10 μM). The purified compounds showing more than 50% displacement of
[3H]SCH23390 at 10 μM were further tested for the affinity (Ki) by using GRAPHPAD PRISM.
Acknowledgements
This work was supported by Center of Excellence Research Program (P01-AT-002038-04,
NCCAM/NIAAA) to D.Y.L.
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