DSpace at VNU: In Vitro Vasoactivity of Zerumbone from Zingiber zerumbet

Original Papers In Vitro Vasoactivity of Zerumbone from Zingiber zerumbet Authors Fabio Fusi 1, Miriam Durante 1, Giampietro Sgaragli 1, Pham Ngoc Khanh 2, Ninh The Son 2, Tran Thu Huong 2, Van Ngoc Huong 3, Nguyen Manh Cuong Affiliations Key words " Zingiber zerumbet l " Zingiberaceae l " zerumbone l " L‑type Ca2+ channel l " whole‑cell patch‑clamp l " vascular smooth muscle l received revised accepted Nov 11, 2014 Dec 24, 2014 January 8, 2015 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 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 37 91 18 12 Fax: + 84 37 56 43 90 nmcuong@inpc.vast.vn Fusi F et al In Vitro Vasoactivity … Dipartimento di Scienze della Vita, Università di Siena, Siena, Italy Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam Faculty of Chemistry, VNU University of Science, Vietnam National University, Hanoi, Vietnam Abstract ! The sesquiterpene zerumbone, isolated from the rhizome of Zingiber zerumbet Sm., besides its widespread use as a food flavouring and appetiser, is also recommended in traditional medicine for the treatment of several ailments It has attracted great attention recently for its effective chemopreventive and therapeutic effects observed 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 Introduction ! Plants, plant extracts, as well as plant-derived products or “phytochemicals” have been used as medicinal aids for millennia as they possess various 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 Malaysia, Laos, Thailand, and Vietnam, where it is distributed mostly in midlands, low mountainous regions, and even in plains [2, 3] Despite its regular uses as a food flavouring and appetiser, the rhizomes of Z zerumbet are also used in traditional medicine as a cure for the treatment of inflammatory- and pain-associated (i.e., oedema, sprain, Planta Med 2015; 81: 298–304 with 30 mM K+ (IC50 values of 16 µM and 102 µM, respectively) In the presence of 60 mM K+, zerumbone concentration-dependently inhibited the contraction induced by the cumulative additions of Ca2+, this inhibition being inversely related to the Ca2+ concentration Phenylephrine-induced contraction was inhibited by the drug, though less efficiently and independently of the presence of an intact endothelium, without affecting Ca2+ release from the intracellular stores Zerumbone inhibited the L-type Ba2+ current (estimated IC50 value of 458.7 µM) and accelerated the kinetics of current decay In conclusion, zerumbone showed an overall weak in vitro vasodilating 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 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 antiinflammatory, antinociceptive, antiulcer, antioxidant, anticancer, antimicrobial, antihyperglycemic, antiallergic, and antiplatelet aggregation activities 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 investigations The essential oil of Z zerumbet rhizome consists mainly of sesquiterpenoids, of which only zerumbone, [(2E,6E,10E)-2,6,9,9-tetramethylcycloundeca-2,6,10-trien-1-one] " Fig 1), the main constituent accounting for the (l 55–85 % of the isolates [3], has been extensively investigated It has been shown to possess in vivo antinociceptive, anti-inflammatory, and antitumour activities, while in vitro it has exhibited antiproliferative and antiplatelet aggregation activities ([2] and references therein) More recently, Batubara et al [5] showed that the inhala- This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 298 Original Papers 299 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 adverse 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 zerumbone, isolated and purified from Z zerumbet rizhomes Results Fig Spasmolytic effect of zerumbone on high K+-induced contraction of rat aorta rings a Rings were depolarised with either 30 or 60 mM extracellular 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 experiments) of the relaxation developed in response to cumulative concentrations (µM) of zerumbone added at the plateau of 30 mM or 60 mM K+-elicited contraction The effect of 100 µM sodium nitroprusside (SNP) is also shown tion 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 animal 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, progression, and metastasis) typically involve the dysregulation of multiple genes and associated cell-signalling pathways [7] Furthermore, owing to the causal relationship existing between inflammation and cancer, zerumbone is receiving increasing interest 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, erroneously safe) make their therapeutic use questionable It is Zerumbone (1) was isolated from the fresh rhizomes of Z zerumbet 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 13 C‑NMR spectrum of compound featured 15 carbon signals assignable to one carbonyl carbon (δC 204.3), three quaternary carbons (δC 137–38), four olefinic methine carbons (δC 160–124), three methylene carbons, and four methyl carbons (δC 42–11) On the basis of these spectroscopic data, compound was identified as the previously reported zerumbone [12] " Fig a, b, zerumbone caused a concentration-deAs shown in l pendent relaxation of rings contracted by 60 mM K+ with an IC50 value of 16 ± 3.2 µM (n = 7) Under the same experimental conditions, the Ca2+ channel blocker nifedipine induced a concentration-dependent spasmolytic activity with an IC50 value 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 zerumbone decreased significantly and its IC50 value (102.0 ± 28.4 µM, n = 6) was much greater than that recorded in rings depolarised " Fig a, b) Zerumbone fully reverted with 60 mM K+ (p < 0.01; l " Fig a) only the 60 mM K+-induced contraction (l 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, " Fig a shows the effects of the sesquiterpene was examined l on the contraction induced by cumulative additions of Ca2+ (0.03–3 mM) to rings depolarised with 60 mM K+ Zerumbone reduced the Ca2+-induced contraction in a concentration-dependent 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 zerumbone, p < 0.05; 8.2 ± 2.2, n = 7, 137.6 µM zerumbone, 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 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 potential pharmacological interaction of zerumbone with (S)-(−)-Bay K " Fig b) In rings pretreated with DMSO, 8644 was assessed (l 10 nM (S)-(−)-Bay K 8644 further stimulated vascular tone by about 36% At the highest concentration assessed, zerumbone also antagonised the stimulating effect of (S)-(−)-Bay K 8644 Fusi F et al In Vitro Vasoactivity … Planta Med 2015; 81: 298–304 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited ! Original Papers Fig Zerumbone inhibition of Ca2+-induced contraction of rat aorta rings depolarised with high K+: effect of (S)-(−)-Bay K 8644 a Ca2+-induced contraction in rings depolarised with a Ca2+-free, 60 mM K+ physiological saline solution in the presence of DMSO or various concentrations of zerumbone 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 Ca2+-induced vascular tone of depolarised 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 The effects of zerumbone on L-type Ba2+ current [IBa(L)] recordings were assessed at a holding potential (Vh) of − 50 mV Zerumbone decreased the current in a concentration-dependent man" Fig a) and, at the maxner (estimated IC50 value of 458.7 µM; l imum concentration tested, significantly decreased the peak inward current in the range between − 20 mV and 50 mV without changing the apparent maximum and the threshold of the cur" Fig b) Under the same experimenrent-voltage relationship (l tal conditions, nifedipine induced a concentration-dependent inhibition of the current with an IC50 value 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 " Fig a) Zerumcould be fitted by a two-exponential function (l bone accelerated the τ of inactivation in a concentration-depen" Fig b) dent manner without affecting the τ of activation (l " Fig a, zerumbone caused a concentration-deAs shown in l 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 recorded on rings with an intact endothelium Under the same ex- Fusi F et al In Vitro Vasoactivity … Planta Med 2015; 81: 298–304 Fig 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 relationships, recorded from a Vh of − 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 perimental conditions, the Ca2+ channel blocker verapamil induced 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 " Fig b, pretreatment with 137.6 µM zerumbone did shown in l 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+ channel 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) This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 300 Fig Effects of zerumbone on IBa(L) current kinetics of single rat tail artery myocytes a Traces of conventional whole-cell IBa(L) elicited with 250ms clamp pulses to 10 mV from a Vh of − 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 (τact) and inactivation (τinact) 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 Dunnettʼs post-test Fig Effect of zerumbone on the phenylephrine-induced contraction of rat aorta rings a Concentration-response curves for zerumbone in endothelium-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 contractions either in the absence (-Ca2+) or in the presence (+ Ca2+) of extracellular Ca2+, 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, Studentʼs t-test for paired samples Discussion ! 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 zerumbet 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 features 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 concentration 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 concentration of K+, as is the case of nifedipine, whose potency increases as the membrane voltage (i.e., the concentration of extracellular 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 stimulated by the Ca2+ channel agonist (S)-(−)-Bay K 8644; this can also be observed with the well-known Ca2+ channel antagonists nifedipine, verapamil, and diltiazem [18] Fourth, zerumbone antagonised IBa(L) in a concentration-dependent manner Taken together, these results identify zerumbone as a novel Ca2+ channel blocker that could be viewed as a potentially useful antihypertensive 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 antagonised IBa(L) at a level and with a potency lower than those found in the inhibition of high K+-induced contractions Therefore, 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] However, vasorelaxation induced by K+ channel openers is inversely related to the extracellular concentration of K+ In fact, the anti- Fusi F et al In Vitro Vasoactivity … Planta Med 2015; 81: 298–304 301 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited Original Papers Original Papers spasmodic effect of the well-known K+ channel opener cromakalim [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+ channels have to be activated in order to respond to Ca2+ antagonist drugs In fact, within the frame of the “state-dependent pharmacology” of the channel, the state-dependent, open channel inhibition 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-inactivated state [22] This hypothesis is supported by the observation 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 observed 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 participation 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+ release from intracellular, phenylephrine-sensitive stores The latter observation also demonstrates that zerumbone did not block α1 adrenergic 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 zerumbone-induced myorelaxation, other mechanisms may play a role, this deserving further investigations However, since its in vitro chemopreventive anticancer activity takes place at concentrations at least one to two orders of magnitude lower [24] than its IC50 as a vasodilator, zerumbone can be considered safe towards vascular effects 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), decapitated, and exsanguinated, as described elsewhere [25] The endothelium 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; ADInstruments) The preparations were allowed to equilibrate for 60 in a modified Krebs-Henseleit saline solution (containing in mM : NaCl 118; KCl 4.75; KH2PO4 1.19; MgSO4 · 7H2O 1.19; NaHCO3 25; 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 otherwise indicated Control preparations were treated with the drug vehicle only Materials and Methods Spasmolytic effect of zerumbone on aorta rings depolarised with high K+ concentrations ! General experimental procedures 13 C NMR (125 MHz), with tetramethylsilane as an internal standard, 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 identified by the ethnobotanist Dr Nguyen Quoc Binh (Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hanoi) A herbarium specimen (MC-355) was deposited in the herbarium of the Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam Fusi F et al In Vitro Vasoactivity … Planta Med 2015; 81: 298–304 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 ginger-fragrant, yellow layer containing volatile oil was removed from the top of the hydrosol, dried over anhydrous Na2SO4, and cooled at °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 Steady tension was evoked in rings by physiological saline solution 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 and then washed for 90 with a Ca2+-free physiological saline solution containing 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 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 302 Original Papers 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 experiment, 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 mM EGTA replaced the physiological saline solution Rings were exposed to this solution for 15 [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 taken as an index of the influx of Ca2+ from the extracellular space triggered in part by the emptied stores and in part by α1-adrenoceptor stimulation Phenylephrine-elicited contractions were obtained 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 artery under the following conditions: the artery was incubated at 37 °C in mL of 0.1 mM Ca2+ external solution (in mM: 130 NaCl, 5.6 KCl, 10 HEPES, 20 glucose, 1.2 MgCl2 · H2O, and Na-pyruvate; pH 7.4) containing 20 mM taurine (prepared by replacing NaCl with equimolar taurine), 1.35 mg/mL collagenase (type XI), mg/mL soybean trypsin inhibitor, and mg/mL BSA, gently bubbled with a 95 % O2/5 % CO2 gas mixture, as previously described [27] Cells, stored in 0.05 mM Ca2+ external solution containing 20 mM taurine and 0.5 mg/mL BSA at °C under normal atmosphere, were used for experiments within two days after isolation [28] The cells were continuously superfused with external solution containing 0.1 mM Ca2+ and 30 mM tetraethylammonium using a peristaltic pump (LKB 2132) at a flow rate of 400 µL/min The conventional whole-cell patch-clamp method [29] was employed to voltage-clamp smooth muscle cells Recording electrodes were pulled from borosilicate glass capillaries (WPI) and firepolished to obtain a pipette resistance of 2–5 MΩ when filled with internal solution [containing in mM: 100 CsCl, 10 HEPES, 11 EGTA, CaCl2 (pCa 8.4), MgCl2 · H2O, Na-pyruvate, succinic acid, oxalacetic acid, Na2-ATP, and phosphocreatine; pH was adjusted to 7.4 with CsOH] An Axopatch 200B patchclamp 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 Vh of − 50 mV and recorded as previously described [25], did not run down during the following 40 [30] The osmolarity of the 30 mM tetraethylammonium- and mM Ba2+-containing external solution (320 mosmol) and that of the internal solution (290 mosmol; [31]) was measured with an osmometer (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 solution until a new steady-state level of current was achieved The fraction of current in the absence of a drug remaining in the presence of each drug concentration was plotted against the drug concentration Chemicals Phenylephrine, acetylcholine, collagenase (type XI), trypsin inhibitor, BSA, tetraethylammonium chloride, EGTA, HEPES, taurine, (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 Haën AG; ryanodine (purity ≥ 98 %) was from Calbiochem Zerumbone (100 mM stock solution), dissolved directly in DMSO, and nifedipine 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 analytical grade and used without further purification Statistical analysis Analysis of data was accomplished by using GraphPad Prism version 5.04 (GraphPad Software, Inc.) Data are reported as mean ± SEM; n is the number of rings or cells processed (indicated in parentheses), isolated from at least three animals Statistical analyses 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 using GraphPad InStat version 3.06 (GraphPad Software) In all comparisons, p < 0.05 was considered significant Zerumbone-mediated relaxations were expressed as a percentage of phenylephrine-, 30 mM or 60 mM K+-mediated contraction 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 obtained 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 Development 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 Fusi F et al In Vitro Vasoactivity … Planta Med 2015; 81: 298–304 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited substance or vehicle was present for 30 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 % 303 Original Papers selected 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 References Newman DJ, Cragg GM Natural products as sources of new drugs over the 30 years from 1981 to 2010 J Nat Prod 2012; 75: 311–335 Yob NJ, Jofrry SM, Affandi MM, The LK, Salleh MZ, Zakaria ZA Zingiber zerumbet (L.) 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680: 22– 27 31 Stansfeld C, Mathie A Recording membrane currents of peripheral neurones in short-term culture In: Wallis DI, editor Electrophysiology A practical approach Oxford: IRL Press; 1993: 3–28 This document was downloaded for personal use only Unauthorized distribution is strictly prohibited 304 Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use  ... Watson LK, Ernst E The use of ginger (Zingiber officinale) for the treatment of pain: a systematic review of clinical trials Pain Med 2011; 12: 1808–1818 Batubara I, Suparto IH, Sadiah S, Matsuoka... causal relationship existing between inflammation and cancer, zerumbone is receiving increasing interest in anticancer drug development programs since it modulates inflammation-related molecular... structure of zerumbone isolated from Z zerumbet Smith b Trace (representative of 3–6 similar experiments) of the relaxation developed in response to cumulative concentrations (µM) of zerumbone added at