Zerumbone was tested for vasorelaxing activity on rat aorta rings and for L-type Ba2+ current blocking activity on single myocytes isolated from the rat-tail artery.. In the presence of
Trang 1!
Plants, plant extracts, as well as plant-derived products or“phytochemicals” have been used as medicinal aids for millennia as they possess vari-ous and significant biological activities [1] Thus, traditionally, they are included in the diet of many human societies (in particular the African and Asian ones) owing to the common belief that they are beneficial to health
Zingiber zerumbet (L.) Smith (Zingiberaceae) (Vietnamese name“Gung gio”) is an up to 1-m tall ginger with pale flowers, fragrant rhizomes, and spear-shape leaves, originating from Southeast Asia It grows in tropical countries including Ma-laysia, Laos, Thailand, and Vietnam, where it is distributed mostly in midlands, low mountainous regions, and even in plains [2, 3] Despite its regu-lar uses as a food flavouring and appetiser, the rhizomes of Z zerumbet are also used in
tradition-al medicine as a cure for the treatment of inflam-matory- and pain-associated (i.e., oedema, sprain,
rheumatism), digestive system (i.e., constipation, diarrhoea), and skin disease-related ailments [2, 4] Various studies, based on a range of in vitro and in vivo model systems, have shown the anti-inflammatory, antinociceptive, antiulcer, antioxi-dant, anticancer, antimicrobial, antihyperglyce-mic, antiallergic, and antiplatelet aggregation ac-tivities of Z zerumbet rhizome (reviewed in [2])
For this reason, of all the parts of the plant, the rhizome has been subjected to broad chemical in-vestigations The essential oil of Z zerumbet rhi-zome consists mainly of sesquiterpenoids, of which only zerumbone, [(2E,6E,10E)-2,6,9,9-tet-ramethylcycloundeca-2,6,10-trien-1-one]
(l"Fig 1), the main constituent accounting for the
55–85% of the isolates [3], has been extensively investigated It has been shown to possess in vivo antinociceptive, anti-inflammatory, and antitu-mour activities, while in vitro it has exhibited antiproliferative and antiplatelet aggregation ac-tivities ([2] and references therein) More re-cently, Batubara et al [5] showed that the
inhala-Abstract
!
The sesquiterpene zerumbone, isolated from the rhizome of Zingiber zerumbet Sm., besides its widespread use as a food flavouring and appe-tiser, is also recommended in traditional medi-cine for the treatment of several ailments It has attracted great attention recently for its effective chemopreventive and therapeutic effects ob-served in various models of cancer To assess the zerumbone safety profile, a pharmacology study designed to flag any potential adverse effect on vasculature was performed Zerumbone was tested for vasorelaxing activity on rat aorta rings and for L-type Ba2+ current blocking activity on single myocytes isolated from the rat-tail artery
The spasmolytic effect of zerumbone was more marked on rings stimulated with 60 mM than
with 30 mM K+(IC50values of 16 µM and 102 µM, respectively) In the presence of 60 mM K+, zer-umbone concentration-dependently inhibited the contraction induced by the cumulative addi-tions of Ca2+, this inhibition being inversely re-lated to the Ca2+concentration Phenylephrine-in-duced contraction was inhibited by the drug, though less efficiently and independently of the presence of an intact endothelium, without af-fecting Ca2+release from the intracellular stores
Zerumbone inhibited the L-type Ba2+current (es-timated IC50value of 458.7 µM) and accelerated the kinetics of current decay In conclusion, zer-umbone showed an overall weak in vitro vasodi-lating activity, partly attributable to the blocking
of the L-type Ca2+channel, which does not seem
to represent, however, a serious threat to its widespread use
In Vitro Vasoactivity of Zerumbone from
Zingiber zerumbet
Huong 3 , Nguyen Manh Cuong 2
2 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam
3 Faculty of Chemistry, VNU University of Science, Vietnam National University, Hanoi, Vietnam
Key words
Bibliography
DOI http://dx.doi.org/
10.1055/s-0034-1396307
Published online February 25,
2015
Planta Med 2015; 81: 298 –304
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032 ‑0943
Correspondence
Dr Fabio Fusi
Università di Siena
Dipartimento di Scienze della
Vita
via A Moro 2
53100 Siena
Italy
Phone: + 39 05 77 23 44 38
Fax: + 39 05 77 23 44 46
fabio.fusi@unisi.it
Correspondence
Assoc Prof Nguyen Manh
Cuong
Institute of Natural Products
Chemistry
Vietnam Academy of Science
and Technology
18 Hoang Quoc Viet Street
122100 Cau Giay, Hanoi
Vietnam
Phone: + 84 4 37 91 18 12
Fax: + 84 4 37 56 43 90
nmcuong@inpc.vast.vn
Trang 2tion of zerumbone increases food consumption and body weight
gain in rats
Zerumbone has attracted great attention recently for its potent
chemopreventive and therapeutic effects In fact, it modulates
an array of important molecular targets (e.g., signal transduction
and apoptotic pathways) in tumour cells in vitro as well as in
ani-mal models of cancer (reviewed in [6]) The fact that zerumbone
is multi-target oriented is a very desirable property for cancer
therapy, as carcinomas at the various stages (i.e., initiation,
pro-gression, and metastasis) typically involve the dysregulation of
multiple genes and associated cell-signalling pathways [7]
Fur-thermore, owing to the causal relationship existing between
in-flammation and cancer, zerumbone is receiving increasing
inter-est in anticancer drug development programs since it modulates
inflammation-related molecular targets [8, 9]
The lack of scientific and clinical data in support of the efficacy
and safety of phytochemicals represents the major encumbrance
to the acceptance of traditional herbal preparations by medical
doctors [10] Moreover, the scarce or null recordings of adverse
reactions to herbal remedies (considered natural and, as such,
er-roneously safe) make their therapeutic use questionable It is
therefore desirable that the safety of these preparations and/or their active principles is established through detailed studies To convince the regulatory committees that zerumbone is safe as well as efficacious, it is necessary to determine its potential ad-verse effects on the cardiovascular system, as part of a Safety Pharmacology “core battery” programme [11] Therefore, the aim of the present study was to assess the vascular activity of zer-umbone, isolated and purified from Z zerumbet rizhomes
Results
!
Zerumbone (1) was isolated from the fresh rhizomes of Z zerum-bet crude extract by steam distillation After recrystallisation three times using absolute EtOH, zerumbone was isolated as white needle crystals with 98 % purity Its molecular formula was found to be C15H22O from the ESI‑MS pseudomolecular peak
at m/z 219.17 429 ([M + H]+) (calcd for C15H23O 219.17 483) The
13C‑NMR spectrum of compound 1 featured 15 carbon signals as-signable to one carbonyl carbon (δC204.3), three quaternary car-bons (δC137–38), four olefinic methine carbons (δC160–124), three methylene carbons, and four methyl carbons (δC42–11)
On the basis of these spectroscopic data, compound 1 was identi-fied as the previously reported zerumbone [12]
As shown inl"Fig 1 a, b, zerumbone caused a concentration-de-pendent relaxation of rings contracted by 60 mM K+with an IC50 value of 16 ± 3.2 µM (n = 7) Under the same experimental condi-tions, the Ca2+channel blocker nifedipine induced a concentra-tion-dependent spasmolytic activity with an IC50value of 8.0 ± 3.4 nM (n = 27) When the rings were depolarised with lower K+
concentrations (i.e., 30 mM), the spasmolytic potency of zerum-bone decreased significantly and its IC50value (102.0 ± 28.4 µM,
n = 6) was much greater than that recorded in rings depolarised with 60 mM K+(p < 0.01;l"Fig 1 a, b) Zerumbone fully reverted only the 60 mM K+-induced contraction (l"Fig 1 a)
To test the hypothesis that zerumbone may compete with Ca2+
within the channel pore, the dependence of its inhibition on the contraction induced by the addition of extracellular Ca2+to high
K+-depolarized rings, in Ca2+-free physiological saline solution, was examined.l"Fig 2 a shows the effects of the sesquiterpene
on the contraction induced by cumulative additions of Ca2+
(0.03–3 mM) to rings depolarised with 60 mM K+ Zerumbone re-duced the Ca2+-induced contraction in a concentration-depen-dent manner (AUC values of 85.0 ± 7.1, n = 17, DMSO; 72.3 ± 18.3,
n = 12, 13.8 µM zerumbone; 41.7 ± 9.5, n = 11, 45.9 µM zerum-bone, p < 0.05; 8.2 ± 2.2, n = 7, 137.6 µM zerumzerum-bone, p < 0.001); a significant reduction in maximum response was also observed It
is also evident that the inhibition exerted by 45.9 µM and 137.6 µM zerumbone, when calculated as a percentage of tension recorded in the presence of DMSO, was inversely related to the extracellular Ca2+ concentration (from 79.3 % and 96.1 % at
300 µM Ca2+ to 35.4 % and 78.1 % at 3 mM Ca2+, respectively)
Under the same experimental conditions, nifedipine induced a concentration-dependent antispasmodic activity with an IC50 value of 27.1 ± 3.1 nM (n = 7)
At the end of the assay, after the last addition of Ca2+, any poten-tial pharmacological interaction of zerumbone with (S)-(−)-Bay K
8644 was assessed (l"Fig 2 b) In rings pretreated with DMSO,
10 nM (S)-(−)-Bay K 8644 further stimulated vascular tone by about 36 % At the highest concentration assessed, zerumbone
al-so antagonised the stimulating effect of (S)-(−)-Bay K 8644
Fig 1 Spasmolytic effect of zerumbone on high K + -induced contraction
of rat aorta rings a Rings were depolarised with either 30 or 60 mM
extra-cellular K + In the ordinate scale, the response is reported as a percentage of
the initial tension induced by 30 or 60 mM K + , taken as 100 % Data points
are mean ± SEM (n = 3–6) Inset: chemical structure of zerumbone isolated
from Z zerumbet Smith b Trace (representative of 3–6 similar
experi-ments) of the relaxation developed in response to cumulative
concentra-tions (µM) of zerumbone added at the plateau of 30 mM or 60 mM K +
-elic-ited contraction The effect of 100 µM sodium nitroprusside (SNP) is also
shown.
Trang 3The effects of zerumbone on L-type Ba2+current [IBa(L)]
record-ings were assessed at a holding potential (Vh) of− 50 mV
Zerum-bone decreased the current in a concentration-dependent
man-ner (estimated IC50value of 458.7 µM;l"Fig 3 a) and, at the
max-imum concentration tested, significantly decreased the peak
in-ward current in the range between− 20 mV and 50 mV without
changing the apparent maximum and the threshold of the
cur-rent-voltage relationship (l"Fig 3 b) Under the same
experimen-tal conditions, nifedipine induced a concentration-dependent
in-hibition of the current with an IC50value of 19.1 ± 5.0 nM (n = 3)
Under control conditions, the current evoked at 10 mV from a Vh
of− 50 mV activated and then declined with a time course that
could be fitted by a two-exponential function (l"Fig 4 a)
Zerum-bone accelerated theτ of inactivation in a
concentration-depen-dent manner without affecting theτ of activation (l"Fig 4 b)
As shown inl"Fig 5 a, zerumbone caused a
concentration-de-pendent relaxation of endothelium-denuded rings contracted by
0.3 µM phenylephrine Zerumbone, however, did not fully revert
the phenylephrine-induced contraction Similar results were
re-corded on rings with an intact endothelium Under the same
ex-perimental conditions, the Ca2+channel blocker verapamil in-duced a concentration-dependent spasmolytic activity with IC50 values of 813.3 ± 329.3 nM (endothelium denuded, n = 6) and 4.3 ± 1.7 µM (endothelium intact, n = 12), respectively
Vasorelaxing agents can antagonise phenylephrine-promoted contractions by inhibiting phenylephrine-induced Ca2+ release from intracellular stores and/or extracellular Ca2+ influx As shown inl"Fig 5 b, pretreatment with 137.6 µM zerumbone did not affect the contraction elicited by 10 µM phenylephrine in
Ca2+-free medium When the normal external Ca2+concentration was restored, with phenylephrine still present, the sesquiterpene significantly inhibited the ensuing contraction Under the same experimental conditions, the sarcoplasmic reticulum Ca2+ chan-nel blocker ryanodine antagonised phenylephrine-induced Ca2+
release from intracellular stores (from 32.4 ± 3.5 % DMSO to 11.1 ± 1.8 %, ryanodine, n = 11; p < 0.001, Studentʼs t-test for paired samples) leaving unaltered the extracellular Ca2+influx (from 74.4 ± 4.8 % to 84.7 ± 1.9 %, n = 11; p > 0.05)
Fig 3 Zerumbone inhibition of IBa(L)of single rat-tail artery myocytes.
a Concentration-dependent effect of zerumbone at the peak of IBa(L)trace.
On the ordinate scale, the response is reported as a percentage of the control Data points are mean ± SEM (n = 4 –5) b Current-voltage relation-ships, recorded from a Vhof − 50 mV, constructed prior to the addition (control) and in the presence of 458.7 µM zerumbone Data points are mean ± SEM (n = 5) * p < 0.05, ** p < 0.01 vs control, Studentʼs t-test for paired samples.
Fig 2 Zerumbone inhibition of Ca 2+ -induced contraction of rat aorta
rings depolarised with high K + : effect of (S)-( −)-Bay K 8644 a Ca 2+ -induced
contraction in rings depolarised with a Ca 2+ -free, 60 mM K + physiological
saline solution in the presence of DMSO or various concentrations of
zer-umbone In the ordinate scale, the response is reported as a percentage of
the initial tension induced by 0.3 µM phenylephrine, taken as 100 % Data
points are mean ± SEM (n = 7 –17) * p < 0.05, *** p < 0.001 vs DMSO.
b Effect of 10 nM (S)-( −)-Bay K 8644 on Ca 2+ -induced vascular tone of
de-polarised rings treated with zerumbone Columns are mean ± SEM (n = 7 –
16) and represent the percentage of the response to 60 mM K + , taken as
100 % * p < 0.05, *** p < 0.001 vs ‑Bay K 8644, Studentʼs t-test for paired
samples; ### p < 0.001 vs DMSO, one-way ordinary ANOVA and Dunnettʼs
post-test.
Trang 4!
To our knowledge, this is the first report of the vascular activity of
zerumbone The present findings demonstrate that the drug is a
weak vasodilating agent targeting plasmalemmal L-type Ca2+
channels that regulate Ca2+influx from the extracellular milieu
Zerumbone was isolated as white needle crystals from Z
zerum-bet rhizomes in a 0.1 % yield by recrystallisation in absolute EtOH,
and its purity, determined by HPLC, reached 98 % (data not
shown)
Myorelaxation promoted by zerumbone shared several basic
fea-tures of the Ca2+channel blockers such as nifedipine [13] First,
the extent of the inhibition of the high K+-induced contraction
by zerumbone was inversely related to the external
concentra-tion of Ca2+[14] Second, this inhibition seemed to depend on
membrane potential [15, 16] In fact, vasorelaxation induced by
Ca2+channel blockers is directly related to the extracellular
con-centration of K+, as is the case of nifedipine, whose potency
in-creases as the membrane voltage (i.e., the concentration of
extra-cellular K+) rises [17] The potency of zerumbone increased as the
external K+ concentration augmented from 30 mM to 60 mM
This finding can be explained by postulating that more positive membrane voltages favour channel blocking by the drug [17]
Third, zerumbone inhibited the Ca2+-induced contraction stimu-lated by the Ca2+channel agonist (S)-(−)-Bay K 8644; this can also
be observed with the well-known Ca2+channel antagonists nife-dipine, verapamil, and diltiazem [18] Fourth, zerumbone an-tagonised IBa(L)in a concentration-dependent manner Taken to-gether, these results identify zerumbone as a novel Ca2+channel blocker that could be viewed as a potentially useful antihyperten-sive agent However, its Ca2+antagonist activity takes place at concentrations at least two to four orders of magnitude higher than the clinically used nifedipine and verapamil, thus devaluing its pharmacological significance Furthermore, zerumbone an-tagonised IBa(L)at a level and with a potency lower than those found in the inhibition of high K+-induced contractions There-fore, other mechanisms beyond Ca2+channel blocking activity might concur to its myorelaxant activity K+channels are known
to play a key role in the maintenance of vessel tone [19] How-ever, vasorelaxation induced by K+channel openers is inversely related to the extracellular concentration of K+ In fact, the
anti-Fig 4 Effects of zerumbone on IBa(L)current kinetics of single rat tail
ar-tery myocytes a Traces of conventional whole-cell IBa(L)elicited with
250-ms clamp pulses to 10 mV from a Vhof − 50 mV, measured in the absence
(control) or presence of various concentrations (µM) of zerumbone Traces
recorded in the presence of zerumbone were magnified so that the peak
amplitude matched that of the control b Time constant for the activation
( τ act ) and inactivation ( τ inact ) measured in the absence (none) or presence
of different concentrations of zerumbone Columns represent mean ± SEM
(n = 5) ** p < 0.01 and *** p < 0.001, repeated measures ANOVA and
Dunnett ʼs post-test.
Fig 5 Effect of zerumbone on the phenylephrine-induced contraction of rat aorta rings a Concentration-response curves for zerumbone in endo-thelium-denuded or ‑intact rings precontracted by 0.3 µM phenylephrine.
In the ordinate scale, relaxation is reported as the percentage of the initial tension induced by phenylephrine, taken as 100 % Data points are mean ± SEM (n = 4–6) b Columns represent 10 µM phenylephrine-induced con-tractions either in the absence (-Ca 2+ ) or in the presence (+ Ca 2+ ) of extra-cellular Ca 2+ , recorded in rings preincubated with vehicle (DMSO) or 137.6 µM zerumbone Columns are mean ± SEM (n = 8) and represent the percentage of the response to 0.3 µM phenylephrine, taken as 100 %.
* p < 0.05 vs DMSO, Student ʼs t-test for paired samples.
Trang 5spasmodic effect of the well-known K+channel opener
cromaka-lim [20] can be observed at depolarisation promoted by 25/
30 mM K+, but not at that promoted by 60 mM K+[21] Therefore,
K+channels were unlikely stimulated by zerumbone
When Ca2+channel inhibition is voltage dependent, Ca2+
chan-nels have to be activated in order to respond to Ca2+antagonist
drugs In fact, within the frame of the“state-dependent
pharma-cology” of the channel, the state-dependent, open channel
inhi-bition that leads to the faster L-type Ca2+channel inactivation
kinetics observed in the presence of zerumbone may explain its
Ca2+channel blocking activity This effect likely originated from
the interaction of zerumbone with the channel in the
voltage-in-activated state [22] This hypothesis is supported by the
observa-tion that the potency of zerumbone was lower in single myocytes
as compared to depolarised rings where inactivated channels
likely predominate, in agreement with what is commonly
ob-served with nifedipine [23]
Findings obtained on aorta rings stimulated with phenylephrine
provided important information on the mechanism of action of
zerumbone This drug, in fact, relaxed both endothelium-intact
and endothelium-denuded rings contracted by phenylephrine
with similar potency and efficacy, thus ruling out the
participa-tion of endothelium-derived vasodilators (e.g., NO) to this effect
Furthermore, zerumbone inhibited the influx of extracellular
Ca2+triggered by phenylephrine while leaving unaffected Ca2+
re-lease from intracellular, phenylephrine-sensitive stores The
lat-ter observation also demonstrates that zerumbone did not block
α1adrenergic receptors, as suggested by its quantitatively similar
antispasmodic and spasmolytic activities
The pharmacological analysis demonstrated that zerumbone was
provided with weak vasodilating effects on rat aorta rings, partly
due to a negative modulation of L-type Ca2+ channel influx
Although K+channel opening activity is unlikely involved in
zer-umbone-induced myorelaxation, other mechanisms may play a
role, this deserving further investigations However, since its in
vitro chemopreventive anticancer activity takes place at
concen-trations at least one to two orders of magnitude lower [24] than
its IC50as a vasodilator, zerumbone can be considered safe
to-wards vascular effects
Materials and Methods
!
General experimental procedures
13C NMR (125 MHz), with tetramethylsilane as an internal
stan-dard, was performed on a Bruker Avance 500 MHz spectrometer,
whereas the HR‑MS analysis was done with a Varian FT‑ESI‑MS
mass spectrometer; column chromatography was carried out on
silica gel (230–400, 400–630 mesh, Merck) Purity of the product
was examined by an HPLC‑MS spectrometer
Plant materials
The rhizomes of Z zerumbet were collected in March 2011 at
mountainous regions in Tamdao, Vinhphuc province, Vietnam
(21°31′N latitude and 105°33′E longitude) The plant was
identi-fied by the ethnobotanist Dr Nguyen Quoc Binh (Vietnam
Na-tional Museum of Nature, Vietnam Academy of Science and
Tech-nology, Hanoi) A herbarium specimen (MC-355) was deposited
in the herbarium of the Institute of Natural Products Chemistry,
VAST, Hanoi, Vietnam
Isolation and purification Fresh rhizomes of Z zerumbet (2.0 kg) were cut into small pieces and distilled by water steam using a Clevenger apparatus over a period of 3–4 h at the boiling water temperature Then the gin-ger-fragrant, yellow layer containing volatile oil was removed from the top of the hydrosol, dried over anhydrous Na2SO4, and cooled at 4 °C overnight The white precipitate was filtered through a G-4 porous glass filter and recrystallised three times using absolute EtOH to obtain zerumbone with a yield of 0.1 %
The purity of zerumbone, determined using an HPLC system, was 98 %
Aorta ring preparation All animal care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals published by the U S National Institutes of Health (NIH Publication No 85–
23, revised 1996) and were approved by the Animal Care and Ethics Committee of the Università di Siena, Italy (08–02–2012)
Aorta rings (2 mm wide), either endothelium-intact or‑denuded, were prepared from male Wistar rats (350–400 g; Charles River Italia), anaesthetised (i p.) with a mixture of Ketavet®(30 mg/kg ketamine; Intervet) and Xilor®(8 mg/kg xylazine; Bio 98), decapi-tated, and exsanguinated, as described elsewhere [25] The endo-thelium was removed by gently rubbing the lumen of the ring with the curved tips of a forceps Each arterial ring was mounted over two rigid parallel, L-shaped stainless steel bars, one fixed in place and the other attached to an isometric transducer (Fort 25, WPI) Contractile tension was recorded with a digital PowerLab data acquisition system (PowerLab 8/30; ADInstruments) and analysed by using LabChart 7.3.7 Pro (Power Lab; ADInstru-ments) The preparations were allowed to equilibrate for 60 min
in a modified Krebs-Henseleit saline solution (containing in
mM : NaCl 118; KCl 4.75; KH2PO41.19; MgSO4· 7H2O 1.19;
NaH-CO325; glucose 11.5; CaCl2· 2H2O 2.5; gassed with a 95 % O2/5 %
CO2 gas mixture to create a pH of 7.4) Endothelium integrity was tested as previously described [25] Experiments were mostly conducted on endothelium-denuded rings unless other-wise indicated Control preparations were treated with the drug vehicle only
Spasmolytic effect of zerumbone on aorta rings depolarised with high K+concentrations Steady tension was evoked in rings by physiological saline solu-tion containing either 30 mM or 60 mM K+(prepared by replacing NaCl with equimolar KCl); cumulative concentration-response curves were constructed with sequential increments of 0.5 log units until a stable state was observed In each arterial ring, only one concentration-response curve was performed At the end of each experiment, 10 µM nifedipine followed by 100 µM sodium nitroprusside were added to test muscle functional integrity
Spasmolysis was evaluated as a percentage of the initial response
to K+, taken as 100 %
Effect of zerumbone on the concentration-response curve for Ca2+
Rings were stimulated with 60 mM K+ for 15 min and then washed for 90 min with a Ca2+-free physiological saline solution containing 1 mM EGTA The preparations were then challenged with 0.3 µM phenylephrine to empty the intracellular Ca2+stores
The zerumbone antispasmogenic response to Ca2+(0.03–3 mM) was assayed on rings depolarised with Ca2+-free 60 mM K+by constructing cumulative concentration-response curves The test
Trang 6substance or vehicle was present for 30 min before as well as
throughout the concentration-response curve procedure At the
end of each experiment, 10 nM (S)-(−)-Bay K 8644 and 100 µM
sodium nitroprusside were added to test L-type Ca2+channels as
well as smooth muscle functional integrity The antispasmodic
effect was evaluated as a percentage of the initial response to
60 mM K+, taken as 100 %
Myorelaxant effect of zerumbone on aorta rings
contracted by phenylephrine
Steady tension was evoked in rings, either endothelium-intact or
‑deprived, by 0.3 µM phenylephrine; thereafter the drug under
investigation was added cumulatively At the end of each
experi-ment, 100 µM sodium nitroprusside was added to test muscle
functional integrity Spasmolysis was evaluated as a percentage
of the initial response to phenylephrine, taken as 100 %
Effect of zerumbone on both Ca2+release from
intracellular stores and extracellular Ca2+influx
triggered by phenylephrine
In order to get insight on the action mechanism of the drug, a
Ca2+-free solution containing 1 mM EGTA replaced the
physiolog-ical saline solution Rings were exposed to this solution for
15 min [26] and then stimulated with 10 µM phenylephrine, the
ensuing contraction being taken as an index of the internal stored
Ca2+ release External Ca2+ (3.5 mM) was then restored in the
presence of phenylephrine, and the ensuing contraction was
tak-en as an index of the influx of Ca2+from the extracellular space
triggered in part by the emptied stores and in part byα1
-adreno-ceptor stimulation Phenylephrine-elicited contractions were
ob-tained after a 30-min incubation with the vehicle alone or with
zerumbone Responses were evaluated as the percentage of the
contraction induced by 0.3 µM phenylephrine in physiological
saline solution, taken as 100 %
Smooth muscle cell isolation procedure and
whole-cell patch clamp recordings
Smooth muscle cells were freshly isolated from the tail main
ar-tery under the following conditions: the arar-tery was incubated at
37 °C in 2 mL of 0.1 mM Ca2+external solution (in mM: 130 NaCl,
5.6 KCl, 10 HEPES, 20 glucose, 1.2 MgCl2· 6 H2O, and 5
Na-pyru-vate; pH 7.4) containing 20 mM taurine (prepared by replacing
NaCl with equimolar taurine), 1.35 mg/mL collagenase (type XI),
1 mg/mL soybean trypsin inhibitor, and 1 mg/mL BSA, gently
bubbled with a 95 % O2/5 % CO2gas mixture, as previously
de-scribed [27] Cells, stored in 0.05 mM Ca2+external solution
con-taining 20 mM taurine and 0.5 mg/mL BSA at 4 °C under normal
atmosphere, were used for experiments within two days after
isolation [28] The cells were continuously superfused with
ex-ternal solution containing 0.1 mM Ca2+and 30 mM
tetraethylam-monium using a peristaltic pump (LKB 2132) at a flow rate of
400 µL/min
The conventional whole-cell patch-clamp method [29] was
em-ployed to voltage-clamp smooth muscle cells Recording
electro-des were pulled from borosilicate glass capillaries (WPI) and
fire-polished to obtain a pipette resistance of 2–5 MΩ when filled
with internal solution [containing in mM: 100 CsCl, 10 HEPES,
11 EGTA, 1 CaCl2(pCa 8.4), 2 MgCl2· 6 H2O, 5 Na-pyruvate, 5
suc-cinic acid, 5 oxalacetic acid, 3 Na2-ATP, and 5 phosphocreatine;
pH was adjusted to 7.4 with CsOH] An Axopatch 200B
patch-clamp amplifier (Molecular Devices Corporation) was used to
generate and apply voltage pulses to the clamped cells and record the corresponding membrane currents
IBa(L), elicited from a Vhof− 50 mV and recorded as previously de-scribed [25], did not run down during the following 40 min [30]
The osmolarity of the 30 mM tetraethylammonium- and 5 mM
Ba2+-containing external solution (320 mosmol) and that of the internal solution (290 mosmol; [31]) was measured with an os-mometer (Osmostat OM 6020, Menarini Diagnostics)
After a steady baseline of current was established, the indicated concentrations of drug were applied to the cell in external solu-tion until a new steady-state level of current was achieved The fraction of current in the absence of a drug remaining in the pres-ence of each drug concentration was plotted against the drug concentration
Chemicals Phenylephrine, acetylcholine, collagenase (type XI), trypsin in-hibitor, BSA, tetraethylammonium chloride, EGTA, HEPES, taur-ine, (S)-(−)-Bay K 8644 (purity ≥ 98%), verapamil (purity ≥ 99%), and nifedipine (purity≥ 98%) were from Sigma Chimica; sodium nitroprusside (purity≥ 99%) was from Riedel-De Hặn AG; rya-nodine (purity ≥ 98%) was from Calbiochem Zerumbone (100 mM stock solution), dissolved directly in DMSO, and nifedi-pine or (S)-(−)-Bay K 8644, dissolved in EtOH, were diluted at least 1000 times prior to use All these solutions were stored at
− 20°C and protected from light by wrapping the containers with aluminium foil The resulting concentrations of DMSO and EtOH (below 0.1 %, v/v) failed to alter the response of the preparations
Phenylephrine was dissolved in 0.1 M HCl Sodium nitroprusside was dissolved in distilled water All other substances were of an-alytical grade and used without further purification
Statistical analysis Analysis of data was accomplished by using GraphPad Prism ver-sion 5.04 (GraphPad Software, Inc.) Data are reported as mean ± SEM; n is the number of rings or cells processed (indicated in par-entheses), isolated from at least three animals Statistical analy-ses and significance as measured by either one-way ordinary or repeated measures ANOVA (followed by Dunnettʼs post-test), or Studentʼs t-test for paired samples (two tailed) were obtained us-ing GraphPad InStat version 3.06 (GraphPad Software) In all comparisons, p < 0.05 was considered significant
Zerumbone-mediated relaxations were expressed as a percent-age of phenylephrine-, 30 mM or 60 mM K+-mediated contrac-tion Data were plotted using the GraphPad Software with the sigmoid curve fitting performed by nonlinear regression; these curves were used to derive the maximal response and the IC50 values
Time constants (τ) of IBa(L)activation and inactivation were ob-tained by a fit from the current value at the beginning to that at the end of the voltage pulse by a two-exponential function using pCLAMP 9.2.1.9 (Molecular Devices Corporation) All fits showed
a correlation coefficient > 0.98
Acknowledgements
!
This work was supported by a grant, No 104.01–2010.25, from the National Foundation for Science and Technology Develop-ment of Vietnam (NAFOSTED) and by the Ministero degli Affari Esteri (Rome, Italy), as stipulated by Law 212 (26–2–1992), to the project“Discovery of novel cardiovascular active agents from
Trang 7selected Vietnamese medicinal plants” We wish to thank Dr M.
Lenoci for assistance with some preliminary experiments
Conflict of Interest
!
The authors declare no conflict of interest
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