Journal of Liquid Chromatography & Related

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ISSN: 1082-6076 (Print) 1520-572X (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc20

RP-HPLC Analysis of an
AlkaloidMethyllycaconitine from Mutagenic
Delphinium malabaricum (Huth) Munz
Firdose R. Kolar, Swaroopa R. Ghatge, Nilesh V. Pawar & Ghansham B. Dixit
To cite this article: Firdose R. Kolar, Swaroopa R. Ghatge, Nilesh V. Pawar & Ghansham B.
Dixit (2015) RP-HPLC Analysis of an AlkaloidMethyllycaconitine from Mutagenic Delphinium
malabaricum (Huth) Munz, Journal of Liquid Chromatography & Related Technologies, 38:20,
1802-1807, DOI: 10.1080/10826076.2015.1110706
To link to this article: http://dx.doi.org/10.1080/10826076.2015.1110706

Accepted author version posted online: 23

Oct 2015.

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Journal of Liquid Chromatography & Related Technologies, 38: 18021807, 2015

Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2015.1110706

RP-HPLC Analysis of an AlkaloidMethyllycaconitine from

Mutagenic Delphinium malabaricum (Huth) Munz
FIRDOSE R. KOLAR, SWAROOPA R. GHATGE, NILESH V. PAWAR, and GHANSHAM B. DIXIT

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Laboratory of Cytogenetics and Plant Breeding, Department of Botany, Shivaji University, Kolhapur, India

A systematic search of some phytochemicals in Delphinium malabaricum will be undertaken to evaluate the therapeutic quality of
D. malabaricum. The populations of D. malabaricum were subjected to analysis of alkaloids by Reversed phase HPLC technique.
Among the various alkaloids present in Delphinium species methyllycaconitine is a principle alkaloid. Therefore, the populations of
D. malabaricum were assessed for Methyllycaconitine (MLA) content in the mutants and their control. The results revealed that the
MLA content was highest with 0.01% concentration of EMS (0.78 mg/g plant material) as compared to the control (0.76 mg/g plant
material). The concentration of MLA decreased with an increase in dose/concentration of the mutagens. Less concentration of MLA in
the roots of D. malabaricum indicates that it is less toxic and a further decrease in concentration after mutagenic treatment minimizes
its toxic effects. As these populations are growing in the same environmental and edaphic conditions, observed variability can be
related to genetic makeup altered due to mutation. The RP-HPLC analysis results could form the basis of a more detailed study of
root alkaloids and any marked similarity/dissimilarity in their alkaloid pattern may draw reasonable conclusions of the difference
between the mutants and the parent cultivar.
Keywords: alkaloids, Delphinium malabaricum, methyllycaconitine, mutation, RP-HPLC, TLC

Introduction

Experimental

Delphinium species are known to be rich sources of diterpenoid

and norditerpenoid alkaloids, in particular, methyllycaconitine
(MLA). Methyllycaconitine, one of the principal norditerpenoid
alkaloid of Delphinium species, possesses curare-like properties
and is used in medical practice.[1,2] It was recently found
that methyllycaconitine and its structural analogs interact with
certain types of nicotinic acetylcholine receptors (nAChRs) of
nerve cells of mammals and insects.[3,4] These compounds
stimulated interest because similar interactions are the focus
of studies on Alzheimers disease, and the search for highly
effective insecticides among derivatives of the alkaloids is also
promising.[5,6] It is known that methyllycaconitine is produced
by plants of the genus Delphinium (larkspur). However, the
study of the alkaloid composition of plant Delphinium
malabaricum growing in the region of Western Ghats of India,
has not received much attention. In this contest, the current
study was intended to quantify the methyllycaconitine content
in D. malabaricum and a comparative study was also performed
among the D. malabaricum and its mutants for methyllycaconitine content in its mutagenic populations, by using RP-HPLC
technique.

Induction of Mutation

Address correspondence to: Firdose R. Kolar, Laboratory of

Cytogenetics and Plant Breeding, Department of Botany, Shivaji University, Kolhapur 416 004 (MS), India. E-mail:firdose.kousar@
gmail.com
Color versions of one or more of the figures in the article can
be found online at www.tandfonline.com/ljlc.

Mutation was induced in Delphinium malabaricum by using

chemical mutagens such as ethyl methane sulfonate (EMS),
sodium azide (SA), and physical mutagen like gamma rays.
Total 300 seeds were used for each concentration/dose of the
treatment and control.
Mutagenic Treatment
EMS and SA Treatment
For chemical mutagen treatment seeds were presoaked for 12 hr
in distilled water, blotted dry and treated with freshly prepared
aqueous solution of ethyl methane sulfonate (EMS) and sodium
azide (SA) at different concentrations (0.01, 0.05, 0.10, 0.15,
0.20, 0.25, and 0.30%) for 6 hr at room temperature (23
2C)
with intermittent shaking. Immediately after the treatment, the
seeds were washed thoroughly with distilled water for 30 min
to leach out residual chemicals.
Gamma Rays Treatment
Dry and healthy seeds of Delphinium malabaricum were
irradiated from a 60CO source at Bhabha Atomic Research
Center (BARC), Mumbai, with a dose of 5, 10, 15, 20, 25,
and 30 kR and with dose rate 2.8 kR per minute and untreated
seeds were used as control.
After mutagenic treatment seeds were sown in the experimental plots within the Botanical Garden, Department of Botany,
Shivaji University, Kolhapur (MS) India. M1 plants were
harvested and grown in successive seasons and developed M2

RP-HPLC Analysis of an AlkaloidMethyllycaconitine

and M3 generations. M2 generation was carefully screened for
various mutations and the mutants scored were bulked and raised
in field to obtain M3 generation. In M3 generation mutant
populations of different mutagenic treatments were assessed
for alkaloid content.

1803
centrifuged at 10,000 rpm for 15 min and the supernatant was
filtered through Whatman filter paper No.1 using Buchner
funnel. All these extracts were stored at 4C in air tight
containers for further use.
Sample Preparation

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Extraction and Estimation of Alkaloids

Preliminary test for the presence of alkaloids in D. malabaricum
was carried out by following the method described by
Harborne.[7] 5 g of the dried sample (Rhizome) was weighed into
a 250-mL beaker and 200 mL of 10% acetic acid in ethanol was
added and covered and allowed to stand for 4 hr. This was filtered
and the extract was concentrated on a water bath to one-quarter of
the original volume. Concentrated ammonium hydroxide was
added dropwise to the extract until the precipitation was complete.
The whole solution was allowed to settle and the precipitate was
collected and washed with dilute ammonium hydroxide and then
filtered. The residue is the alkaloid, which was dried and weighed.
The amount of alkaloid was calculated using the formula:
Total Alkaloids% The weight of alkaloid residue (X)=
Weight of sample (W)  100

For RP-HPLC analysis the extracted solutions were filtered

through a 0.22-m nylon syringe filter. The filtered solutions
were then used for RP-HPLC analysis. All these extracts were
kept at 4C and were used for the RP-HPLC analysis.
Instrumentation
The RP-HPLC system consisted of two pumps (model 515
Waters, Milford, MA, USA), a manual injector (Rheodyne,
Cotati, CA, USA), and an UV-VIS detector (Waters 2487).
The instrument was controlled by the Empower 2 software
(Waters) for data acquisition and processing. Chromatographic
separation was achieved on a C-18 reversed phase column
(Princeton SPHER, 5 , 250 mm) kept at room temperature.
Chromatographic Conditions

where X is the weight of the residue; Y is the weight of the filter

paper; and Z is the weight of the filter paper alkaloid residue.

A mobile phase consisting of 0.2 M aqueous ammonium acetate

adjusted to the required pH (5.0) with formic acid. Acetonitrile

TotalX Z  Y
Screening of Alkaloids by Thin Layer Chromatography
Thus, the crude extract of alkaloids obtained was weighed and
dissolved in a known volume of ethanol and was used for
TLC. For this technique precoated TLC aluminum sheets
(20  20 cm Silica gel 60 F254) were used. The alkaloid spots
were separated using the solvent mixture chloroform and methanol (15:1). The color and Rf values of the separated alkaloids
were recorded both under ultraviolet (UV 254 nm) and visible
light after spraying with Dragendorffs reagent.
RP-HPLC Analysis of AlkaloidMethyllycaconitine
For the RPHPLC analysis of alkaloid Methyllycaconitine in D.
malabaricum and its mutant populations, mutant rhizomes
obtained from treatments of all the concentrations of mutagens
from M3 generation along with control were selected.
Extract Preparation
Rhizomes of D. malabaricum from different concentrations of all
the mutagenic treatments including control were obtained from
the field and washed thoroughly under tap water and then rinsed
with double-distilled water. Rhizomes were blotted to dry with
tissue towel and dried in an oven at 60C. After drying, rhizomes
were crushed separately and finely ground to a powder in an
electric blender. The one gram powder of rhizome from each
treatment was extracted using 10 mL ethanol with continuous
shaking on a rotary shaker for 24 hr. The mixture was then

Fig. 1. TLC profile of alkaloids extracted from the roots of

Delphinium malabaricum by Harborne method (1973); the
elucidated spots were visualized as orange spots after spraying with
Dragendorff's reagent. (a) TLC plate exposed to day light, (b) TLC
plate exposed to UV 254 nm. 1-3: Different concentration of
extracts (1:25 l, 2: 50 l, 3:100 l).

1804
Results

Prior to the RP-HPLC analysis the preliminary phytochemical

screening and quantitative estimation of the chemical constituents of the D. malabaricum was made. The results of the study
showed that the roots were rich in alkaloids as determined by

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was used as organic modifier. The mobile phase was pumped at

a flow rate of 1 mL min 1 in the proportion of 70:30, respectively, and the detection wavelength was used 270 nm. The
20 L of the sample was injected using Hamilton syringe. The
program was run up to 20 min. The spectral data were collected
and analyzed.

F. R. Kolar et al.

Fig. 2. Reverse-phase HPLC chromatograms of alkaloid profiles of Delphinium malabaricum: a: Chromatogram of MLA (125 ppm); b:
Control; c: 0.01% EMS; d: 0.05% EMS; e: 0.10% EMS; f: 0.15% EMS; g: 0.20% EMS; h: 0.25% EMS; i: 0.30% EMS; j: 0.01% SA.

RP-HPLC Analysis of an AlkaloidMethyllycaconitine

layer chromatography for their further confirmation. During

chromatographic separation, it was observed that alkaloids appeared as orange color bands when sprayed with Dragendorffs
reagent (Figure 1). The results of the TLC studies demonstrated
the presence of 10 different types of alkaloids with 10 different
Rf values with range from 0.06
0.005 to 0.61
0.01 indicating

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the method described by Harborne.[7] The root extracts of

D. malabaricum contained the highest percentage crude
yield of alkaloids (3.13% or 31.3 mg/g dry weight). Since the
preliminary phytochemical screening showed the presence of
a considerable amount of alkaloids, which are the major phytoconstituents, there is double necessity to perform thin

1805

Fig. 3. Reverse-phase HPLC chromatograms of alkaloid profiles of Delphinium malabaricum: a: 0.05% SA; b: 0.10% SA; c: 0.15% SA;
d: 0.20% SA; e: 0.25% SA; f: 0.30% SA; g: 5kR gamma rays; h: 10kR gamma rays; i: 15kR gamma rays; j: 20kR gamma rays.

1806

F. R. Kolar et al.

the presence of different types of alkaloids in D. malabaricum.

However, the bands of alkaloids developed on the TLC plate
were not identified but the result of the study authenticates and
confirms the presence of several alkaloids in D. malabaricum.

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RP-HPLC Analysis of AlkaloidMethyllycaconitine

The alkaloids seen in the different fractions during TLC analyses
have been successfully separated and determined by RP-HPLC.
Chromatograms that show separation in different fractions of D.
malabaricum and standard compound methyllycaconitine are
shown in Figures 2 and 3. For RP-HPLC analysis we have used
only one reference compound, that is, Methyllycaconitine, which
is a principle alkaloid found in Delphinium species. Methyllycaconitine (MLA) is a diterpenoid alkaloid found in many species of
Delphinium.[8,9] The objective of the present study was to conduct
an analysis of the levels of MLA in D. malabaricum and its
mutants. Thus to determine the effect of mutagens on MLA
content, the roots of D. malabaricum were analyzed for the presence of MLA by RP-HPLC using an authentic standard of Methyllycaconitine. The details of the HPLC elution profile of D.
malabaricum alkaloids in control and its mutants were presented
in Table 1 and their chromatograms were shown in Figures 2
and 3. The results specified that the extracts contained several
compounds including methyllycaconitine (retention time:
11.793 min) and many unknown compounds (Figures 2 and 3).
The distribution of MLA in each of the 19 samples was analyzed
with respect to the relative concentration of the selected alkaloid as
Table 1. RP-HPLC Quantification of Alkaloid-Methyllycaconitine
in D. malabaricum and its mutants
HPLC
Dose/ Retention
Conc. of time
Concentration
Percentage2
Sample Mutagen mutagen (min)
(mg/g)1
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

Control
EMS

SA

Gamma
Rays

0.01%
0.05%
0.10%
0.15%
0.20%
0.25%
0.30%
0.01%
0.05%
0.10%
0.15%
0.20%
0.25%
0.30%
5 kR

11.91
12.21
12.02
11.97
12.06
12.15
11.99
12.11
11.84
11.83
11.94
11.75
12.02
12.10
12.03
11.83

0.765
0.320
0.397
0.788
0.320
0.272
0.200
0.196
0.365
0.492
0.430
0.370
0.363
0.359
0.239
0.266

0.076
0.032
0.039
0.078
0.032
0.027
0.020
0.019
0.036
0.049
0.043
0.037
0.036
0.035
0.023
0.026

10 kR
15 kR
20 kR

11.94
12.00
12.08

0.328
0.397
0.312

0.032
0.039
0.031

1
Results are expressed as milligram of fraction equivalent (Methyllycaconitine)
per gram of sample.
2
Percentage Relative % of total concentration.

determined from the chromatogram of methyllycaconitine; the

total MLA alkaloid concentration was variable with a range from
0.196 mg/g to 0.788 mg/g of dry weight (Table 1). The concentration of MLA decreased with an increase in dose/concentration
of the mutagen. The highest concentration of MLA was recorded
in the control (0.76 mg/g), which decreased to 0.196 mg/g at 0.3%
EMS. However, the concentration of MLA was increased to some
extent at 0.1% EMS (0.78 mg/g) than the control. Less concentration of MLA in the roots of D. malabaricum indicates that it
is less toxic and a further decrease in concentration after mutagenic
treatment minimizes its toxic effects. However, the data obtained
indicated a possibility in the alteration of gene functions for the
synthesis of Methyllycaconitine.

Discussion
Delphinium species are well known as a source of alkaloids of
pharmaceutical importance. Alkaloids, as a rule, do not occur
singly in plants. The plant usually produces a series of alkaloids,
which may differ only slightly in physical and chemical characters. In the plants, alkaloids may be systemic, that is, distributed
throughout, or restricted to specific organs like roots (Aconite,
Belladona), rhizomes and roots (Ipecac, Hydrastis), stem barks
(Cinchona, Pomegranate), leaves (Hyoscyamus, Belladona),
fruits (Pepper, Conium), or seeds (Nuxvomica, Areca).[10] In
present study, the analysis of alkaloids using thin layer chromatographic technique showed the presence of various alkaloids in
D. malabaricum. TLC is the principle technique of chromatography. Therefore, further analysis by higher techniques such
as high performance liquid chromatography (HPLC) or NMR
(nuclear magnetic resonance) spectroscopy for clear identification of alkaloids present in D. malabaricum is necessary.
Alkaloid synthesis pathways are highly regulated and involve
controls at the cell, tissue, plant, and environmental levels.[11]
Benn and May[12] indicated that larkspur alkaloids are synthesized
in roots, but little is known about further synthesis and translocation. Nicotinic acetylcholine receptors (nAchRs) are a family
of ligand gated ion channels that are widely distributed in the
human brain.[13] These receptors have numerous receptor subtypes
composed of combinations of a2-7, a9-10, and b2-4subunits.[14,15]
This subunit composition represents a major factor in determining
pharmacological and functional properties of these receptors.
NAchRs are involved in a number of physiological and behavioral
conditions; hence, there is a pressing need for subtype selective
agonists and antagonists to elucidate the biological roles of these
receptors and to provide candidates for drug discovery. The a7
nAChR subtype is among the most prevalent in the brain and
has been implicated as playing a key role in conditions such as
schizophrenia, Alzheimers disease, and epilepsy. Methyllycaconitine (MLA) is one of only a few compounds (including the peptide toxins, a-bungarotoxin, and a-conotoxin) that bind with high
affinity and selectivity to the a7nAChR. MLA is therefore a prime
lead compound for development of new therapies targeting the
a7nAChR. Based on its unique interaction with nAChRs, methyllycaconitine is an inspiration source for medicinal chemistry. We
have therefore embarked on a program to isolate MLA from D.
malabaricum. Methyllycaconitine (MLA) is one member of a
larger family of diterpene alkaloids, isolated from Delphinium
and Aconitum species of plants that have been known as sources

RP-HPLC Analysis of an AlkaloidMethyllycaconitine

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of poisons and medicinal agents for a long time.[16,17] As it was

mentioned previously, methyllycaconitine is the most potent nonpeptide competitive antagonist of nicotinic acetylcholine receptors
known with selectivity for a7 nAChR subtype at low nanomolar
concentrations (1.7 nM).[1823] The results discussed in the present
study revealed that the MLA content was less in D. malabaricum
(0.76 mg/g plant material) as compared to other Delphinium species. The concentration of MLA in D. malabaricum decreased
after mutagenic treatment. Less concentration of MLA in the roots
of D. malabaricum indicates that it is less toxic and a further
decrease in concentration after mutagenic treatment minimizes
its toxic effects and makes it suitable for its use in the drug industry. Methyllycaconitine is the main lead compound of the research
presented in this study; therefore, a more detailed discussion on
this compound and its role in drug development will be presented.

Conclusions
The Delphinium species contain a number of alkaloids of pharmaceutical importance in addition to the alkaloid methyllycaconitine
that need to be assessed when considering the therapeutic value of
the plant. The extremely potent and selective nicotinic acetylcholine receptor antagonist methyllycaconitine alkaloids are finding
increasing use as neurochemical probes and as targets for structure-activity relationship statistics. In this work, an assay procedure for MLA, which utilizes reverse-phase HPLC with UV
absorbance detection at 270 nm, is described. The current method
has shown acceptable precision and adequate sensitivity for the
quantification of MLA. The method was successfully applied to
quantify the concentrations of MLA in D. malabaricum. Thus, it
can be concluded that reverse-phase HPLC method is adequate
to detect and quantify the alkaloids. The reverse-phase separation
may be preferred due to readily available columns, reduced solvent
use, and simplicity of the electrospray ionization source. The
method can thus be applied both to the measurement of MLA in
crude plant extracts and to isolated alkaloid samples in order to
determine the precise levels of any trace contamination with this
highly potent alkaloid prior to bioassay. Further studies are needed
to find other toxic and nontoxic alkaloids and to determine the toxicity and therapeutic potential of Delphinium malabaricum.

Acknowledgments
Authors are grateful to the Head, Department of Botany, Shivaji
University, Kolhapur. We are also thankful to Prof. N. S. Desai
and Mr. Subash Kudale for their extensive help. The financial
support received with gratitude from the University Grant
commission, New Delhi, is most appreciated.

Funding
The work was supported by University Grant commission, New
Delhi.

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