eCommons@AKU
Department of Biological & Biomedical Sciences
Medical College, Pakistan
August 2011
Studies on the Chemical Composition and
Possible Mechanisms Underlying the
Antispasmodic and Bronchodilatory Activities of
the Essential Oil of Artemisia maritima L.
Abdul Jabbar Shah
Aga Khan University
Anwarul-Hassan Gilani
Aga Khan University
Kanza Abbas
Aga Khan University
Munawwer Rasheed
Amir Ahmed
See next page for additional authors
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Recommended Citation
Shah, A., Gilani, A., Abbas, K., Rasheed, M., Ahmed, A., Ahmad, V. (2011). Studies on the Chemical Composition and Possible
Mechanisms Underlying the Antispasmodic and Bronchodilatory Activities of the Essential Oil of Artemisia maritima L.. Archives of
Pharmacal Research, 34(8), 1227-1238.
Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/137
Authors
Abdul Jabbar Shah, Anwarul-Hassan Gilani, Kanza Abbas, Munawwer Rasheed, Amir Ahmed, and Viqar
Uddin Ahmad
This article is available at eCommons@AKU: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/137
Arch Pharm Res Vol 34, No 8, 1227-1238, 2011
DOI 10.1007/s12272-011-0801-0
Studies on the Chemical Composition and Possible Mechanisms
Underlying the Antispasmodic and Bronchodilatory Activities of the
Essential Oil of Artemisia maritima L.
Abdul Jabbar Shah1,2, Anwarul-Hassan Gilani1, Kanza Abbas1, Munawwer Rasheed3, Amir Ahmed4, and
Viqar Uddin Ahmad5
1
Drug Discovery and Natural Products Research Unit, Department of Biological and Biomedical Sciences, Aga Khan University Medical College, Karachi 74800, Pakistan, 2Department of Pharmacy, COMSATS, Institute of Information Technology, Abbottabad 22060, Pakistan, 3Department of Chemistry, University of Karachi, Karachi 75270, Pakistan, 4Pharmaceutical Research Center, Pakistan Council for Scientific and Industrial Research, Karachi Laboratory Complex, Karachi
75270, Pakistan, and 5HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of
Karachi, Karachi 75270, Pakistan
(Received July 21, 2010/Revised November 8, 2010/Accepted December 16, 2010)
This study describes the chemical composition of the essential oil of Artemisia maritima
(Am.Oil) and the pharmacological basis for its medicinal use in gut and airways disorders.
Twenty five compounds, composing 93.7% of the oil, were identified; among these, chrysanthenyl
propionate and elixene were identified for the first time from any Artemisia species. The Am.Oil
(0.3-1.0 mg/mL) suppressed spontaneous and high K+ (80 mM)-induced contractions in isolated
rabbit jejunum, suggestive of an antispasmodic effect mediated possibly through calcium channel blockade. The calcium channel blockade activity was confirmed when pre-treatment of the
tissue with Am.Oil (0.01-0.03 mg/mL) shifted the Ca++ concentration-response curves to the
right, similar to verapamil and papaverine. In isolated tracheal strips, Am.Oil inhibited carbachol (CCh; 1 µM)-induced contractions more than that induced by K+ and shifted the isoprenaline-induced inhibitory CRCs to the left, similar to papaverine, suggestive of potentiation, while,
verapamil was more potent against K+ than CCh-induced contractions and had no potentiating
effect on isoprenaline-induced inhibitory CRCs. These data indicate that the Am.Oil exhibited
spasmolytic and bronchodilator activities mediated possibly through dual blockade of calcium
channels and phosphodiesterase, which provides the pharmacological basis to the medicinal use
of Artemisia maritima in colic, diarrhea and possibly asthma.
Key words: Artemisia maritima L., Essential oil, Antispasmodic, Bronchodilator, Calcium
antagonist, PDE inhibition, GC-MS, 13C-NMR
INTRODUCTION
Artemisia maritima L. (syn. Artemisia brevifolia
Wall ex DC) belongs to the family Asteraceae. It is a
Correspondence to: Anwarul-Hassan Gilani, Department of Biological and Biomedical Sciences, Aga Khan University Medical
College, Karachi 74800, Pakistan
Tel: 92-21-486-4571, Fax: 92-21-493-4294, 2095
E-mail: [email protected]
Viqar Uddin Ahmad, HEJ Research Institute of Chemistry,
International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan
Tel: 92-21-481-9020, Fax: 92-21-481-9019
E-mail: [email protected]
tall, aromatic perennial herb, commonly known as
seaworm wood (Pullaiah, 2006), as it can tolerate
maritime exposure and often found in salt marshes of
the British Isles, France, Hungary, South Russia,
Siberia, Afghanistan and China. In Pakistan these
plants grow in the Chitral and Swat regions as well as
the mountainous region of Baluchistan (Nasir and Ali,
1972; Nadkarni, 1976). It possesses similar biological
properties as the other wormwoods but is less studied
because of its milder action and potency (Tomova,
1962; Koichi et al., 1965; Munishwar et al., 1967;
Mathela et al., 1994; Eva et al., 1995; Jaitak et al.,
2008). Important constituents of the essential oil from
1227
1228
A. maritima L. has a cajput oil and camphor-like odor
and is reported to contain cineole, thujone, camphene,
a lactone, santonin, which are responsible for the
anthelmintic properties (Trease and Evan, 1985).
Different parts of the plant have been used for medicinal purposes; leaves are administered in nervous
and spasmodic afflictions like asthma, abdominal pain
(antispasmodic) and CNS diseases. The seeds have
been used as an appetizer, expectorant, aphrodisiac, a
cure for indigestion, diarrhoea, and scorpion stings
and is useful for toothaches, ophthalmia, inflammation (Gorsi and Miraj, 2002), a cardiac stimulant and
tonic (Kapoor, 1990).
The pharmacological studies on the essential oil of
the plant are scarce except for a hepatoprotective
(Janbaz and Gilani, 1995) effect by our group and the
plant has not been evaluated pharmacologically for its
use in gastrointestinal and airways disorders. This
study reports the chemical composition of the essential oil of A. maritima and its biological activities on
the gut and airways along with the underlying mechanism for its antispasmodic and bronchodilator activities. The oil potentially works through a dual blockade
of calcium channels and phosphodiesterase thus providing the scientific basis for the medicinal use of the
plant in colic, diarrhea, cough and asthma.
MATERIALS AND METHODS
Essential oil
Fresh, authentic essential oil from A. maritima L.
was purchased from the local market of the Quetta
district, in September 2007.
Instrumentation and identification
The protocol used in chemical studies was similar as
discussed earlier (Gilani et al., 2009). Gas chromatography using FID, was carried out on a Shimadzu gas
chromatograph GC-17A hooked with Shimadzu Class
GC-10 software and equipped with a less polar capillary column SPB-5® (30 m × 0.53 mm ID × 0.50 mm
filter thickness). The analyses were performed with
an initial temperature at 50oC for 5 min and then
ramped at 3oC/min to a final temperature of 210oC
with a run time of 45 min. Injector with splitting ratio
was 1:50 and FID was set at 300oC. Carrier and make
up gas was nitrogen with a flow of 28 and 40 mL/min
at a pressure of 1.0 and 2.5 kg/cm2, respectively. Kovats
retention indices were also calculated (Kovats, 1958).
For GC-EIMS experiments, a Hewlett-Packard 5890
gas chromatograph was combined with a Jeol, JMSHX 110 mass spectrometer operating in EI mode with
standard conditions. Injector was set at 270oC with
A. J. Shah et al.
splitting ratio 1:30. Analyses were performed on the
aforementioned program on an equivalent column
HP-5® (25 m × 0.22 mm and 0.25 mm film thickness).
Mass spectral survey was performed using MS-libraries
(NIST Mass Spectral Search Program, 1998 and 2005).
13
C-NMR (BB and DEPT) spectra of the essential oil
were recorded in CDCl3 on a Bruker Aspect 3000 AM300 spectrometer operating at 75 MHz.
Drugs and standards
The following reference chemicals were obtained
from the sources specified: acetylcholine chloride, carbamyl choline chloride (carbachol), isoprenaline hydrochloride, papaverine hydrochloride and verapamil
(Sigma Chemical Company). All chemicals used were
of the highest purity grade. Stock solutions of all
chemicals were made in distilled water and the dilutions were made fresh on the day of the experiment.
The essential oil was solubilized in 10% DMSO and
dilutions were made in normal saline or distilled
water.
Animals
Experiments performed complied with the guidelines of the Institute of Laboratory Animal Resources,
Commission on Life Sciences, National Research
Council (NRC, 1996). Local rabbits (1.5-2 kg) and
guinea-pigs (500-550 g) of either sex used in the study
were bred and housed in the animal house of The Aga
Khan University under a controlled environment (2325ºC). Animals were given tap water ad libitum and a
standard diet.
Isolated tissue preparations
Rabbit jejunum
The isolated tissue experiments were carried out as
previously described (Ghayur et al., 2007). The animals
had free access to water but were fasted for 24 h
before the experiment. The animals were sacrificed by
cervical dislocation, the abdomen was cut open and
the jejunal portion was isolated. Preparations (2 cm
long) were mounted in 10 mL tissue baths containing
Tyrode’s solution maintained at 37oC and aerated
with a mixture of 5% carbon dioxide in oxygen (carbogen). The composition of Tyrode’s solution (pH 7.4),
in mM, was: KCl 2.7, NaCl 136.9, MgCl2 1.1, NaHCO3
11.9, NaH2PO4 0.4, glucose 5.6 and CaCl2 1.8. A preload of 1 g was applied and the tissues were kept undisturbed for 30 min as an equilibrium period after
which control responses to a sub-maximal dose of
acetylcholine (0.3 mM) were obtained and the tissue
was presumed stable only after the reproducibility of
the said responses.
Chemistry and Antispasmodic Effect of Essential Oil from Artemisia maritima
Under these experimental conditions, rabbit jejunum
exhibits spontaneous rhythmic contractions, allowing
testing the relaxant (spasmolytic) activity directly
without the use of an agonist (Gilani et al., 1994).
Determination of calcium channel blocking
activity
To assess whether the spasmolytic activity of the
test substances, was through calcium channel blockade, K+, as KCl, was used to depolarize the preparations (Farre et al., 1991). K+ (80 mM) was added to
the tissue bath, which produced a sustained contraction. Test substances were then added in a cumulative
fashion to obtain concentration-dependent inhibitory
responses (van Rossum, 1963). The relaxation of intestinal preparations was expressed as percent of the
control response mediated by K+.
To confirm the calcium channel blocking (CCB)
activity of the test substances, tissues were allowed to
stabilize in normal Tyrode’s solution and then replaced with Ca++-free Tyrode’s solution containing
EDTA (0.1 mM) for 30 min in order to remove calcium
from the tissues. This solution was further replaced
with K+-rich and Ca++-free Tyrode's solution, having
the following composition in mM: KCl 50, NaCl 91.04,
MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, glucose
5.55 and EDTA 0.1. Following an incubation period of
30 min, control concentration-response curves (CRCs)
of Ca++ were obtained. When the CRCs of Ca++ were
found super-imposable (usually after two cycles), the
tissue was pre-treated with the test materials for 60
min to test the possible calcium channel blocking
effect. The CRCs of Ca++ were reconstructed in the presence of different concentrations of the test materials.
Guinea-pig trachea
The protocol of Boskabady and Aslani (2006), was
used with some modifications (Gilani et al., 2005b):
trachea was dissected and cut longitudinally on the
ventral side, the rings were opened into strips, which
were then mounted in a 20 mL tissue bath containing
Kreb’s solution, maintained at 37oC and aerated with
carbogen. The composition of Kreb’s solution (pH 7.4)
was (mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl
4.7, KH2PO4 1.3, MgSO4 1.2 and glucose 11.7. A tension
of 1 g was applied to each of the tracheal strips and
was kept constant throughout the experiment. The
tissues were equilibrated for 1 h before the addition of
any drug. Carbachol (CCh; 1 µM) and K+ (80 mM)
were used to induce sustained contractions then test
materials were added cumulatively and the relaxant
activity was expressed as percent of CCh- and K+induced contractions.
1229
Determination of phosphodiesterase inhibitory activity
Guinea-pig tracheal strips were suspended in
normal Kreb’s solution in 20 mL organ baths and
control isoprenaline (0.003-1 µM) inhibitory CRCs
were constructed against CCh-induced contractions as
described earlier (Gilani et al., 2005a). When control
CRCs of isoprenaline were found super-imposable
(usually after two cycles), the tissue was pre-treated
with the test substances for 30 min to test the possible
potentiating effect. The CRCs of isoprenaline were
reconstructed in the presence of different concentrations of test material.
Guinea-pig atria
Spontaneously beating paired atria from guinea-pig
were mounted in 20 mL tissue baths containing normal Kreb’s solution maintained at 32oC and aerated
with carbogen, as described previously (Ghayur and
Gilani, 2006). After an equilibrium period of 30 min,
control responses to isoprenaline (1 µM) and ACh (1
µM) were obtained at least in duplicate. The test substances were then added cumulatively and the effect
on force and rate of contractions were determined as a
percent of the pre-treated control. Tension changes in
the tissue were recorded via a force-displacement transducer (model FT-03) using a Grass model 7 Polygraph.
Statistics
All the data expressed are mean ± S.E.M., and the
median effective concentrations (EC50 values) are
given with 95% confidence intervals (CI). A student ttest with a level of significance of p < 0.05 was used to
determined whether responses were significantly
different (GraphPAD program, GraphPAD). Concentration-response curves were analyzed by non-linear
regression (GraphPAD).
RESULTS AND DISCUSSION
For the chemical analysis, the essential oil of A.
maritima was subjected to GC-FID and GC-EIMS
analysis and the components were characterized
mainly by mass spectral survey (NIST Mass Spectral
Search Program, 1998 and 2005) resulting in the identification of twenty four constituents in the essential
oil, which were further supported by comparing the
retention indices (Kovats, 1958; Vandendool and Kratz,
1963) calculated with those cited in the literature
(Table I and Fig. 1). Similarly, comparison of the 13CNMR spectra of the mixture with those recorded for
the pure authentic compounds in literature further
authenticated the identification. The RI values and
1230
A. J. Shah et al.
Table I. Composition of Essential Oil from Artemisia maritima L.
Constituents
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
α-Pinene
Camphene
β-Pinene
2,3-Dehydro-1,8-cineole
α-Terpinene
1,8-Cineole
Santolina alcohol
γ-Terpinene
β-Thujone
Chrysanthenone
Some p-mentha-dien-ol
L-(-)-Camphor
L-Borneol
Terpinen-4-ol
α-Terpineol
Z-Chrysanthenyl acetate
Bornyl acetate
α-Terpinyl acetate
Z-Chrysanthenyl propionate
E-geranyl acetate
β-Caryophyllene
Aromadendrene
Elixene
Cis-Davanone
Artemone
RI obs
0935
0954
0978
1008
1035
1046
1056
1065
1130
1137
1148
1158
1190
1205
1221
1295
1297
1358
1384
1407
1432
1466
1505
1581
1603
% age
2.34
4.59
2.90
0.84
0.61
41.14
0.79
1.10
1.11
2.80
0.10
20.320
1.66
2.11
1.93
1.65
0.24
0.30
0.29
0.46
4.19
0.22
0.44
1.36
0.23
93.720
Identification
a, b, c
Ref.
d, e
MS, RI
, C-13
MS, RI a, h, C-13 d, e
MS, RI a, b, c, C-13 d, e
MS, RI h
MS, RI a, b, c, C-13 d, e
MS, RI a, b, c, C-13 d, e
MS, RI j
MS, RI a, b, C-13 e
MS, RI a, b, C-13 e
MS, RI p
MS tentative
MS, RI b, h, C-13 e
MS, RI b, h, C-13 e
MS, RI a, c, C-13 e
MS, RI a, b, C-13 e
MS, RI b, r , C-13.s
MS, RI a, c, h, C-13.e
MS, RI a, b, C-13.e
MS, RRT s, C-13.s
MS, RI c, C-13 e
MS, RI b, c, C-13 d, e
MS, RI a, b, C-13 e
MS, RI a
MS, RI b, t, C-13 u
MS w
f, g
R
Rf
R f, g
Ri
Ri
R f, g, i
NR* k, l
Ri
R g, i, m, n
Ri
R f, g, i
Ri
NR* b, q
R f, i
Ri
Rf
NR* b
NR
NR* q
R f, i
NR* b, q
NR
NR b, q, t, v
NR* d
MS: mass spectroscopy, RI: retention index, C-13: C-13 spectroscopy, R: reported, NR: not reported from any species of
Genus Artemisia, NR*: not reported from Artemisia maritima L. References: a) Cardeal et al., 2006, b) Judžentienë and
Buzelytë, 2006, c) Gilani et al., 2009, d) Rahman and Viqar, 1992, e) Kubeczka and Formacek, 2002, f) Munishwar et al.,
1967, g) Koichi et al., 1965, h) Pino et al., 2005, i) Jaitak et al., 2008, j) Tellez et al., 1999, k) Hudaib and Aburjai, 2006, l)
Näf-Müller et al., 1981, m) Tomova, 1962, n) Mathela et al., 1994, o) Formisano et al., 2007, p) Coleman et al., 2007, q)
Flamini et al., 2003, r) Darriet et al., 2009, s) Bail et al., 2008, t) Thomas et al., 1974, u) Sadeghpour et al., 2004, v) Naegeli
et al., 1970.
chemical shift values obtained for the compounds
were in good agreement with the reported data. The
results from qualitative and quantitative analysis of
the essential oil are summarized in Table I.
Genus Artemisia is well studied for the essential oils
but the literature lacks the systemic studies on the
essential oil from the A. maritima L. of Baluchistan
(Pakistan) origin. This genera contains santonin, a
potent vermicide, as one of the main components of
the dried flower (Shah and Mathela, 2006a, 2006b)
while the aerial parts of A. maritima L. are rich in
various types of ketones (Saxena and Jain, 2002). In
the essential oils, one or two of the constituents are
generally dominant in one sample, but lacking in
others (Shah and Mathela, 2006a, 2006b). For
example, the essential oil of A. maritima L. is reported
to contain 1,8-cineol as the major constituent with
chrysanthenone in one collection while borneol and
camphor were the major components in another
collection (Jaitak et al., 2008). The current study also
revealed 1,8-cineol as a major constituent (41.14%)
but followed by camphor (20.32%), camphene (4.59%)
and β-caryophyllene (4.19%) while chrysanthenone and
borneol were observed in relatively in low concentrations. α-Thujone followed by β-thujone have been reported as the major constituents in various species
of Artemisia including A. maritima (Tomova, 1962;
Mathela et al., 1994; Shah and Mathela, 2006b), αthujone was not detected in the current study while βthujone was present (1.11%). Eva et al. (1995) reported
linalool as the major constituent which was not
detectable in this essential oil. It can be called 1,8cineole/camphor chemotype with reference to the work
published for various species of the Artemisia (Başer
Chemistry and Antispasmodic Effect of Essential Oil from Artemisia maritima
1231
Fig. 1. Chemical composition of essential oil from A. maritime L.
and Demirci, 2007; Jaitak et al., 2008).
Irregular monoterpenes in low concentrations were
also observed in A. maritima as reported earlier from
A. herba-alba (Tahar and Tarak, 2006) and A. vulgaris
(Näf-Müller et al., 1981). Z-chrysanthenyl propionate
was identified as a new component from this genus.
MS of this compound resembled that of Z-chrysanthenyl acetate except that a peak of 57 also appears.
The propionate esters of sabinyl, isobornyl and linalyl
alcohol were previously reported from A. maritima.
Elixene is another new component from A. maritima
(Jaitak et al., 2008). The MS of components observed
at RI 1581 and 1603 were identical with cis-davanone.
With the literature value of RI 1586 (Judzentiene and
Buzelyte, 2006) and 1588 (Bail et al., 2008), the 1581
component was considered to be cis-davanone. The
other davanone isomer was concluded to be artemone
as the MS of the artemone is identical to cis-davanone
(Naegeli et al., 1970). Seven components including
santolina alcohol, terpinen-4-ol, a-terpinyl acetate, Egeranyl acetate, aromadendrene, cis-davanone and
artemone have not been previously reported from A.
maritima L., although these are reported in various
other species of the Artemisia (Table I). Altogether
93.7% of the composition was identified.
In line with the potential medicinal use of the plant
in hyperactive gut states, such as colic, Am.Oil was
tested in an isolated rabbit jejunum preparation where
it suppressed the spontaneous contractions (Fig. 2),
with EC50 value of 0.24 mg/mL (0.11-0.39), thus, showing
intestinal smooth muscle relaxation (spasmolytic)
activity (Fig. 2A), similar to that of verapamil and
papaverine (Fig. 2).
The contraction of smooth muscle preparations in-
1232
cluding rabbit jejunum is dependent upon an increase
in the cytoplasmic free [Ca++], which activates the
contractile elements (Karaki and Weiss, 1988). The
increase in intracellular Ca++ occurs either via influx
through voltage-dependant Ca++ channels (VDCs) or
the release of Ca++ from intracellular stores in the
sarcoplasmic reticulum. Periodic depolarization and
repolarization regulates the spontaneous movements
of the intestine and at the height of depolarization,
the action potential appears as a rapid influx of Ca++
via VDCs (Brading, 1981). The inhibitory effect of the
essential oil on spontaneous movements, similar to
verapamil, suggest that the inhibitory effect may be
due to interference either with the Ca++ release or
with the Ca++ influx mediated through VDCs, in addition to other mechanism(s).
We previously observed that the spasmolytic constituents present in different medicinal plant extracts
mediate their effect usually through a CCB effect
(Gilani et al., 1994, 2005b, 2009). To see whether the
spasmolytic effect of the essential oil observed in this
study is also mediated through CCB, a high concentration of K+ (80 mM) was introduced to depolarize the
tissue. The oil was then added in a cumulative fashion
where it caused a concentration-dependent relaxation
of the induced contractions with an EC50 value of 0.37
Fig. 2. Tracings of concentration-dependent inhibitory effects
of (A) essential oil of A. maritima (Am.Oil), (B) papaverine
and (C) verapamil on the spontaneous contractions in isolated rabbit jejunum preparations.
A. J. Shah et al.
mg/mL (0.21-0.62), suggesting CCB effect (Fig. 3A). A
similar effect was observed with papaverine (Fig. 3B),
while verapamil was comparatively more potent against
high K+ precontractions, a typical characteristic of
Ca++ antagonists (Godfraind et al., 1986).
The presence of CCB activity was further confirmed
when pretreatment of the jejunal preparation with
Am.Oil (0.1-0.3 mg/mL) caused a rightward shift in
the Ca++ CRCs (Fig. 2D), similarly to that observed
with verapamil (Fig. 3F) and papaverine (Fig. 3E).
The presence of CCB constituents in Am.Oil provides
the pharmacological basis of the medicinal use for A.
maritima in gastrointestinal disorders, such as colic
and possibly diarrhoea because CCBs are known to be
antispasmodic and antidiarrhoeal (Brunton, 1996).
The non-specific inhibitory pattern of both spontaneous and high K+-induced contractions by Am.Oil
identical to papaverine, a dual inhibitor of Ca++ influx
(Boselli et al., 1998) and PDE (Boswell-Smith et al.,
2006), indicates the possible presence of PDE-like
inhibitory constituent(s), in addition to CCB, particularly when CCBs alone exhibit a certain degree of
selectivity against K+-induced contractions as observed
in this study and reported earlier (Gilani et al., 2005a).
Based on the reported therapeutic potential of CCBs
and PDE inhibitors in airways disorders, particularly
asthma and cough (Mathewson, 1985; Triggle, 1992)
and the known medicinal value of this plant in asthma,
it was further studied for its bronchodilatory effect.
When tested against CCh (1 µM) and high K+-induced
contractions in isolated guinea-pig tracheal strips,
Am.Oil caused a concentration-dependent inhibition
of both precontractions, with EC50 values of 0.41 (0.230.52) and 0.52 mg/mL (0.33-0.81), respectively (Fig.
4A). This was similar to papaverine, with respective
EC50 values of 3.05 (2.25-6.23) and 2.89 µM (3.01-5.36)
(Fig. 4C), suggestive of non-specific tracheal relaxation.
Unlike the essential oil and papaverine, verapamil
was found to be relatively more potent against high
K+-induced contractions (Fig. 4E), as it is a Ca++ antagonist (Farre et al., 1991). The PDE inhibitory activity was further confirmed when pretreatment of tracheal preparations with Am.Oil (0.03-0.1 mg/mL) shifted the isoprenaline-induced inhibitory CRCs to the
left thus showing a potentiating effect (Fig. 4B). This
was similar to papaverine (Fig. 4D), while verapamil
was found to be without a potentiating effect (Fig. 3F).
Cyclic nucleotides, such as cGMP and cAMP play an
important role in the regulation of tracheal tone (Chu,
1984). The β-adrenergic receptor agonist, isoproterenol, induces the relaxation of airways by stimulating β2-adrenoceptors and the formation of cAMP via
activation of adenylate cyclase. Agents that inhibit
Chemistry and Antispasmodic Effect of Essential Oil from Artemisia maritima
1233
Fig. 3. Concentration-dependent inhibitory effects on spontaneous and high K+-induced contractions by (A) essential oil of
A. maritima (Am.Oil), (B) papaverine and (C) verapamil in isolated rabbit jejunum preparations. Figs. 2D, E and F show
the effect on Ca++ concentration-response curves in the absence and presence of increasing concentrations of Am.Oil,
papaverine and verapamil in isolated rabbit jejunum preparations. Values shown are mean ± S.E.M. (n = 3-5).
cyclic nucleotide PDE exert tracheal relaxant and
bronchodilator activities (Chu, 1984), causing relaxation by decreasing the Ca++ oscillation frequency of
airway smooth muscle (Bai and Sanderson, 2006).
Inhibition of PDE activity has been recognized as a
mechanism by which various compounds induce smooth
muscle relaxation (Lohmann et al., 1977; Ward et al.,
1993) and PDE inhibitors are known to be effective
against early and late phase asthmatic responses
(Karlsson, 1996).
The usefulness of PDE inhibitors in asthma is well
established (Teixeira et al., 1997). Interestingly, the
CCBs have also been shown to be useful in cough and
asthma (Mathewson, 1985; Kamei and Kasuya, 1992).
Thus, the co-existence of Ca++ and PDE inhibitory
constituents in the essential oil of A. maritima might
be responsible for its medicinal use in the gut and
airways disorders.
The PDE inhibitors when used alone as a bronchodilator are known to cause cardiac stimulation as a
side-effect (Raeburn et al., 1993); CCBs, on the other
hand are known to cause relaxation of smooth muscle
as well as cardiac depression. To see the outcome of
the combined effect of CCB and PDE inhibition on the
heart, the essential oil was studied in the cardiac preparations. In isolated spontaneously beating guineapig atria (Fig. 5B), Am.Oil caused suppression of force
and rate at distinctly higher concentrations, with
respective EC50 values of 3.47 (1.92-6.30) and 2.29 mg/
mL (1.46-6.30). This was similar to papaverine, which
also caused inhibition of both force and rate at high
concentrations with respective EC50 values of 47.0
(29.17-75.75) and 21.28 µM (15.05-28.85) (Fig. 5B).
However, verapamil caused inhibition of both force
and rate at similar concentration (0.01-1.0 µM) as in
smooth muscle (Fig. 5C). It is likely that the distinctly
high dose required for the cardiac depressant effect of
the essential oil, is due to by interference of PDE inhibitory constituents, which are known to be stimulants
in the heart (Brain and Hoffman, 2001; Orallo et al.,
2005), a pattern similar to that observed with papaverine. Thus, the co-existence of PDE inhibitory and
CCB constituents in the essential oil is likely to offset
the cardiac side-effects and offering added advantage
when used for gut and airways hyperactivity disorders.
This type of combination has also been observed in the
1234
A. J. Shah et al.
Fig. 4. Concentration-dependent inhibitory effects of (A) essential oil of A. maritima (Am.Oil), (C) papaverine and (E)
verapamil on CCh- and high K+-induced contractions. Figs. B, D and F show the potentiating effect on the inhibitory
concentration-response curves of isoprenaline against CCh-induced contractions in the absence and presence of different
concentrations of Am.Oil, papaverine and verapamil in isolated guinea-pig tracheal preparations. Values shown are mean ±
S.E.M. (n = 4-6).
essential oil of Nepeta cataria L (Gilani et al., 2009)
and is in line with the concept that the plant extracts,
which combines multiple chemical constituents contain
combinations with synergistic and/or side effect neutralizing potential (Gilani and Atta-ur-Rahman, 2005).
We also observed a dual combination of antispasmodic and bronchodilator activities in another plant
(Artemisia vulgaris) from the same genus (Khan and
Gilani, 2009) but it possessed antimuscarinic activity
instead of phosphodiesterase inhibitory activity, while
both shared the Ca++ antagonist activity. When comparing their phytochemical constituents, A. vulgaris
was found to be well studied with the presence of number of chemicals, such as, adenine, amyrin, artemisiketone, borneol, cadinenol, coumarin, fernenol, pathulenol, stigmasterol, tau-remisin, tetracosanol, thujone,
vulgarin, vulgarol, vulgarole, umbelliferone, inulin,
linalool, muurolol, myrcene, nerol, molybdenum, pinene,
esculin, esculetin and scopoletin, in addition to some
well known compounds such as quercetin, and βsitosterol present in different plants (Nadkarni, 1976;
Ikhsanova et al., 1986; Duke, 1992). These two plants
share a few constituents including borneol, pinone
and thujone but none of these constituents are known
to posses Ca++ channel blocking activity.
Additionally, most of the constituents present in A.
maritima have been reported from other medicinal
plants, such as, Thymus vulgaris L., Thymus tosevii
L., Mentha spicata L., and Mentha piperita L. (Sokovic
et al., 2009), Daucus carota L. (Maxia et al., 2009),
Pycnostachys abyssinica and Pycnostachys eminii
(Hussien et al., 2010), Centaurea ensiformis (Ugur et
al., 2009), etc. The biological activities of some of the
constituents reported are either antibacterial and/or
antioxidants. The chemicals, which possess antibacterial activity, are α-pinene (Sokovic et al., 2010) and
thujone (Laciar et al., 2009), while camphor is an
antioxidant (Sokmen et al., 2004). However, one of the
constituents in A. maritima (aromadendrene) is reported as spasmolytic (Perez-Hernandez et al., 2009)
with unknown underlying mechanism(s). We propose
that this constituent is most probably amongst the
other constituents responsible for the observed antispasmodic effect.
In summary, 2 new compounds among the 24 known
compounds were isolated for the first time from the
essential oil of A. maritima. Pharmacological studies
on different isolated tissues preparations revealed the
presence of spasmolytic and tracheal relaxant activities mediated possibly through dual inhibition of
Ca++ influx and PDE, which provides sound pharmacological evidence in support of its medicinal use in
Chemistry and Antispasmodic Effect of Essential Oil from Artemisia maritima
Fig. 5. Concentration-dependent inhibitory effects of (A)
essential oil of A. maritima (Am.Oil), (B) papaverine and (C)
verapamil on force and rate of atrial contractions in isolated
spontaneously beating guinea-pig atrial preparations. Values
shown are mean ± S.E.M. (n = 4-5).
colic, cough and asthma.
ACKNOWLEDGEMENTS
The authors wish to express their deep sense of
gratitude to the Higher Education Commission Sector
H-9, Islamabad for financial support through the
Distinguished National Professorship allowance.
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