Reisen und Gesundheit Prophylaxe Vorbeugung ist die wichtigste Schutzmaßnahme vor Infektionskrankheiten. Die wichtigsten vorbeugenden Maßnahmen aus Sicht der Tropenmedizin finden Sie hier zusammengestellt. Wozu Prophylaxe? Vorbeugen ist besser als Heilen. Das gilt auch bei Reisen in ferne Länder. Die Vorbeugung richtet sich dabeim nach Reiseziel und Aufenthaltsdauer, nach Reisestil, Jahreszeit und individuellen Bedürfnissen.
Anticonvulsant activity of bisabolene sesquiterpenoids of curcuma longa in zebrafish and mouse seizure models
Contents lists available at Epilepsy & Behavior Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebraﬁsh and mouse seizure models Adriana Monserrath Orellana-Paucar , Ann-Sophie K. Serruys Tatiana Afrikanova , Jan Maes ,Wim De Borggraeve Jo Alen Fabián León-Tamariz Isabel María Wilches-Arizábala ,Alexander D. Crawford Peter A.M. de Witte , Camila V. Esguerra a Laboratory for Molecular Biodiscovery, Department of Pharmaceutical & Pharmacological Sciences, University of Leuven, Leuven, Belgiumb Escuela de Bioquímica y Farmacia, Facultad de Ciencias Químicas, Universidad de Cuenca, Cuenca, Ecuadorc Molecular Design and Synthesis, Department of Chemistry, University of Leuven, Leuven, Belgium Turmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional medicine for the Received 20 December 2011 treatment of epilepsy. To date, in vivo studies on the anticonvulsant activity of turmeric have focused on its Revised 16 February 2012 principal curcuminoid, curcumin. However, poor absorption and rapid metabolism have limited the thera- Accepted 21 February 2012 peutic application of curcumin in humans. To explore the therapeutic potential of turmeric for epilepsy fur- Available online 5 April 2012 ther, we analyzed its anticonvulsant activity in a larval zebraﬁsh seizure assay. Initial experiments revealedthat the anticonvulsant activity of turmeric in zebraﬁsh larvae cannot be explained solely by the effects of Keywords:Curcuma longa curcumin. Zebraﬁsh bioassay-guided fractionation of turmeric identiﬁed bisabolene sesquiterpenoids as ad- ditional anticonvulsants that inhibit PTZ-induced seizures in both zebraﬁsh and mice. Here, we present the ﬁrst report of the anticonvulsant properties of bisabolene sesquiterpenoids and provide evidence which war- rants further investigation toward the mechanistic understanding of their neuromodulatory activity.
2012 Elsevier Inc. All rights reserved.
Ar-turmeroneα-AtlantonePentylenetetrazoleZebraﬁsh PTZ modelMouse PTZ model depression, cognitive impairment and the fact that seizures inabout one third of patients suffering from epilepsy remain resistant Epilepsy is a widespread neurological disorder that affects approx- to these existing treatments Hence, there is a clear need to con- imately 50 million people worldwide . According to the World tinue to identify novel AEDs that effectively control pharmaco- Health Organization (WHO), about 1% of the total burden of disease resistant seizures with minimal or no adverse effects.
corresponds to different forms of epilepsy. Its pharmacological treat- Numerous studies point to medicinal plants as an interesting ment comprises a number of currently available antiepileptic drugs source of novel AEDs . One interesting example in this regard is (AEDs). The main problems concerning AEDs are the high incidence losigamone derived from the kava kava plant and originally used by of side effects ranging from gastrointestinal distress, hepatotoxicity, traditional healers in the South Paciﬁc as an anxiolytic, which isnow in early clinical development as a novel AED . Likewise,Curcuma longa, a perennial herb of the Zingiberaceae family native Abbreviations: AEDs, antiepileptic drugs; DAD, diode array detection; DMSO, di- to South Asia, has been used not only as a condiment and color addi- methyl sulfoxide; dpf, days post-fertilization; ddH2O, double-distilled water; ESI-MS, tive in food but also in traditional medicine against seizures . The electrospray ionization mass spectrometry; i.v., intravenous; i.p., intraperitoneal; LC– major active chemical constituents of turmeric (C. longa rhizome MS, liquid chromatography–mass spectroscopy; MES, maximal electroshock seizure; powder) are the curcuminoids (3–5%) and turmeric oil (2–7%). Tur- MTC, maximum tolerated concentration; PEG, polyethylene glycol; p.o., "per os", orally;PTZ, pentylenetetrazole; RP-HPLC, reverse phase-high performance liquid chromatog- meric oil is mainly composed of bisabolene sesquiterpenoids: ar-, raphy; s.c., subcutaneous; TLE, temporal lobe epilepsy.
α,β-turmerone and α-atlantone, whereas the curcuminoids include ⁎ Corresponding author at: Laboratory for Molecular Biodiscovery, Department of curcumin, monodemethoxycurcumin and bisdemethoxycurcumin Pharmaceutical and Pharmacological Sciences, University of Leuven, Gasthuisberg Nearly all investigations on the medicinal properties of turmeric Campus, Building O&N2, Room 08.4226, Herestraat 49, P.O. Box 824, BE-3000 Leuven, have been focused on curcumin, and its anticonvulsant activity has Belgium. Fax: + 32 16 323460.
E-mail address: (P.A.M. de Witte).
been demonstrated in several rodent models such as the iron- 1525-5050/$ – see front matter 2012 Elsevier Inc. All rights reserved.
doi: A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 induced epileptogenesis maximal electroshock kainic acid- A "voucher specimen" serves as a reliable reference for the identi- induced and pentylenetetrazole-kindling models. Although ty of botanical samples for further investigations. For this reason, a its anticonvulsant properties have been demonstrated, phase I clinical voucher specimen of turmeric (no. 7850) was deposited in the trials have revealed important pharmacokinetic limitations for curcu- Herbarium Azuay (HA), Universidad del Azuay, Cuenca, Ecuador.
min. When administrated p.o., curcumin is poorly absorbed throughthe gut. Therefore, the amount of curcumin in the circulation is very 2.3. Experimental animals low. Thus, pharmacokinetic issues have limited its therapeutic appli-cations. For this reason, formulation studies have been performed to All procedures for animal experiments were performed in enhance curcumin bioavailability The fact that nearly all the accordance with European and National Regulations and ap- studies have been conducted on curcumin has led to the assumption proved by the Animal Care and Use Committee of the University of that the anticonvulsant properties of turmeric are due solely to its ac- Leuven (Belgium).
tivity. However, turmeric oil could also be accountable for its anticon-vulsant activity as other essential oils have been shown to be effective 2.3.1. Zebraﬁsh (D. rerio) in controlling seizure generation Intriguingly, a few studies Adult zebraﬁsh of the Tg (ﬂi 1a: EGFP)y1 strain were reared at have reported on the neuroprotective activity of turmeric oil 28.5 °C on a 14/10-hour light/dark cycle. Eggs were collected from In this paper, we report on the anticonvulsant activity of turmeric natural breeding and fostered in embryo medium (17 mM NaCl, methanolic extract and its constituents as assessed in a zebraﬁsh PTZ- 2 mM KCl, 1.8 mM Ca(NO3)2, 0.12 mM MgSO4, 1.5 mM HEPES buffer induced seizure model. Further conﬁrmation of this activity was car- pH 7.1–7.3 and 0.6 μM methylene blue) in an incubator at 28.5 °C.
ried out in the mouse PTZ model.
Zebraﬁsh are considered ‘embryos' between 0 and 72 hours post- The zebraﬁsh (Danio rerio) has emerged over the last decade as a fertilization (hpf). From 72 hpf to 14 days post-fertilization (dpf), valuable model for genetic studies and drug screening. The strength they are referred to as ‘early larvae' or ‘larvae' Sorting of of this in vivo model relies on its high genetic, physiologic and phar- zebraﬁsh embryos and larvae and medium refreshment were macologic homology to humans. Their high fecundity and small size performed daily until 7 dpf. After completing the experiments, all allow for the performance of tests in a medium- to high-throughput larvae were sacriﬁced through administration of an overdose of fashion using minute (microgram scale) quantities of compound . The zebraﬁsh also holds promise as an in vivo model for iden-tifying novel neuroactive compounds since the dopaminergic, seroto- 2.3.2. Mice (Mus musculus) nergic, and GABAergic systems develop early during embryogenesis Male C57Bl/6 mice (20–30 g) at 8 weeks of age were acquired from and are already functional in larvae . Additionally, their rapid de- Charles River Laboratories. The mice were housed in poly-acrylic cages velopment ex utero and optical transparency make it possible to easily under 12/12-hour light/dark cycle at 28 °C in a quiet room. The detect morphological and behavioral effects of test compounds on liv- animals were fed ad libitum with a pellet diet and water until they ing embryos and larvae . More recently, zebraﬁsh have also prov- were 10 weeks old (the age when all assays were carried out).
en useful for the primary screening of potential novel anticonvulsantsbased on the proconvulsant pentylenetetrazole (PTZ) . Like- 2.4. Preparation of methanolic extract of turmeric wise, PTZ has been used as a chemoconvulsant in rodent models,and the timed PTZ infusion test has proven competence in enabling Turmeric (5 g) was extracted through maceration in methanol the identiﬁcation of AEDs with different mechanisms of action (50 ml). The extract was concentrated using a rotary evaporator In this study, we conﬁrmed the reported anticonvulsant properties (Büchi Rotavapor R-114, Germany) to obtain a yield of 0.20 g.
of turmeric and curcumin. Further testing of turmeric oil and its chro-matographic fractions revealed additional constituents capable of 2.5. Preparation of turmeric volatile oil and isolation of major suppressing PTZ-induced seizure behaviors in larval zebraﬁsh. Mass spectrometry and NMR analyses of these active puriﬁed fractionsrevealed them to belong to the class of bisabolene sesquiterpenoids: Volatile oil from turmeric was obtained by subjecting 4 × 100 g ar-turmerone, α,β-turmerone and α-atlantone. The anticonvulsant of turmeric to hydro-distillation using a Clevenger-type apparatus activity of turmeric oil, ar-turmerone and α,β-turmerone, identiﬁed for 3 h. A pale yellowish and odiferous oil was obtained (8.56 g).
using the zebraﬁsh PTZ assay, was then conﬁrmed in the equivalent Turmeric oil was dried over anhydrous sodium sulfate and stored at mouse PTZ-induced seizure model.
4 °C until used.
The isolation of some major compounds present in the essential 2. Materials and methods oil was carried out by semi-preparative RP-HPLC, as adapted fromthe work of He and colleagues A LaChrom Elite HPLC System 2.1. Chemicals and reagents (VWR Hitachi) equipped with diode array detection (DAD) and anEconosphere 10-μm C18 (250×10 mm) reversed phase column Dimethyl sulfoxide (99.9%, spectroscopy grade) and the curcumi- (Grace Davison Discovery Sciences, Belgium) attached to an noid mixture from turmeric (98%; curcumin, demethoxycurcumin Econosphere 10 μm C18 (33×7 mm) guard column (Grace Davison and bisdemethoxycurcumin) were procured from Acros Organics.
Discovery Sciences, Belgium) were used. Amounts of 10-mg volatile Diethyl ether (99.9%, spectroscopy grade), deuterated chloroform oil were repeatedly processed. The column was operated at a ﬂow (99.8% D, contains 0.1% (v/v) TMS) and PTZ were obtained from rate of 5 ml/min at room temperature. The proﬁle of the gradient elu- Sigma-Aldrich, methanol (99.8%, reagent grade) and acetonitrile tion was: double-distilled water (ddH2O) (A) and acetonitrile (B); (100%, HPLC grade) from Fisher Scientiﬁc. Sodium valproate was 0–15 min, 40–60% B; 15–20 min, 60–100% B. The analytes were moni- obtained from Sanoﬁ-Synthelabo.
tored with DAD at 260 nm. Eight fractions from turmeric oil were indi-vidually collected (). Solvents from the collected fractions were 2.2. Plant material removed by separation between diethyl ether and ddH2O. The etherphase was collected and dried over anhydrous sodium sulfate. Ether Dried rhizome powder of C. longa (turmeric) was acquired from a was removed by passing a slow stream of nitrogen over the sample at local supplier in Belgium with India as the source of origin. Micro- room temperature. The concentrated samples were stored at 4 °C until scopic authentication according to was completed by R. Ansaloni.
A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 larva per well. Each stock solution of sample (dissolved in 100% Fractions collected from the RP-HPLC analysis of DMSO) was diluted in embryo medium to achieve a ﬁnal DMSO con- turmeric oil. The collected fractions are referred centration of 1%. In cases where sample had to be diluted further (i.e.
as percentage (%) of content in turmeric oil.
more than 1:100), DMSO was added so as to maintain a ﬁnal concen- Fraction no.
tration of 1%. Each sample was loaded in duplicate adjacent rows. The larvae were then incubated at room temperature under dark and quiet conditions for 1 h. Equal volumes (100 μl) of embryo medium (vehicle) and 40-mM PTZ were then added respectively to the ﬁrst and second rows of each sample group (resulting in a doubling of the total incubation volume and halving of the ﬁnal DMSO concentra- tion to 0.5% and PTZ to 20 mM). The total locomotor activity of each larva was video-tracked and assessed in the presence of either vehicle Fraction 4: ar-turmerone; fraction 5: α,β-turmerone; fraction 6: α-atlantone.
2.6. Chemical structure elucidation of bisabolene sesquiterpenoids 2.6.1. Nuclear magnetic resonance (NMR) analysis 1H and 13C NMR spectra of fractions 4, 5 and 6 (see below) were obtained from Bruker 300 Avance and Bruker 600 Avance II+ equip- ment using deuterated chloroform as solvent and tetramethylsilane (TMS) as internal standard.
2.6.2. Mass spectroscopy (MS) analysis Total movement in 30 min
The LC–MS analysis was performed on an Agilent 1100 system equipped with degasser, quaternary pump, auto-sampler, UV-DAD detector and thermostated column module coupled to Agilent 6110 3.1 µg/mL
6.2 µg/mL 12.5 µg/mL
single–quadrupole MS. Data acquisition and quantiﬁcation wereobtained from Agilent LC/MSD Chemstation software. Fractions 4, 5 3.1 µg/mL + PTZ
6.2 µg/mL + PTZ
12.5 µg/mL + PTZ
and 6 (see further) were analyzed on a Grace Prevail RP-C18 column 3 μm (150 mm × 2.1 mm) at a ﬂow rate of 0.2 ml/min. The LC gradient comprised two solvents: double-distilled water (ddH2O) + 0.1% for-mic acid (A) and acetonitrile (B); 0–17 min, 40–60% B; 17–32 min,60–100% B.
The ESI-MS analysis was completed in a Thermo Electron LCQ Ad- vantage apparatus with Agilent 1100 pump and injection system coupled to Xcalibur data analysis software.
2.7. Determination of maximum tolerated concentration in zebraﬁsh Total movement in 30 min
The aim of this assay was to determine the range of appropriate 2.5 µg/mL
concentrations to be tested in zebraﬁsh for the evaluation of anticon-vulsant activity. Seven-dpf larvae were placed into a 24-well plate 5 µg/mL + PTZ
2.5 µg/mL + PTZ
10 µg/mL + PTZ
(tissue culture plate, ﬂat bottom, FALCON®, USA), six larvae per well. They were incubated with different concentrations of sample dissolved in 1 ml of embryo medium (at a ﬁnal DMSO concentrationof 1%). The larvae were examined during a period of 24 h in sampleand compared to control group to detect the following signs of toxic- ity: absence of startle response to plate taps, changes in heart rate orcirculation, presence of edema, loss of posture, paralysis and death.
Thus, the maximum tolerated concentration (MTC) was deﬁned as the highest concentration at which no signs of toxicity were observedin 6 out of 6 zebraﬁsh larvae within 24 h of exposure to sample.
Total movement in 30 min
2.8. Evaluation of anticonvulsant activity in the zebraﬁsh PTZ model Zebraﬁsh larvae from 7 dpf were monitored using the ViewPoint 2.5 µg/mL
VideoTrack System for Zebraﬁsh™ (Version 22.214.171.124, ViewPoint, 5 µg/mL + PTZ
2.5 µg/mL + PTZ
10 µg/mL + PTZ
France). The system consists of an infrared light source, a high-resolution digital video camera to capture larval movements within Fig. 1. Evaluation of the anticonvulsant activity of turmeric in the zebraﬁsh PTZ seizure a deﬁned time period (30 min in our experimental set-up) and the assay. (A) Turmeric methanolic extract; (B) curcuminoids and (C) turmeric oil. Tested software to analyze larval locomotor activity.
concentrations are indicated along the x-axis, and the total gross locomotor activityexhibited by zebraﬁsh larvae within 30 min is displayed along the y-axis. Data are The highest concentrations of the samples tested correspond to expressed as the mean ± SD (n = 10–12). Statistically signiﬁcant differences between the previously determined MTC values. Zebraﬁsh larvae were placed vehicle-treated and sample-treated (white bars) or PTZ-treated and sample plus PTZ- in a tissue culture, ﬂat bottom 96-well plate (FALCON®, USA); one treated groups (gray bars) are labeled as * for p b0.05 and ** for p b 0.01.
A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 only, vehicle with sample, PTZ only, or PTZ and sample (see Supple- glass syringes containing: a) sample; heparin 2 UI/ml and b) PTZ mentary Fig. S1). Video-tracking of larval movements commenced (7.5 mg/ml ddH2O; heparin 2 UI/ml). These syringes were mounted exactly 5 min after addition of embryo medium or PTZ to the wells on a motor-driven infusion double pump (ALADOIN-1000 11VDC, and was recorded for 30 min. A total of 8 wells in each plate were 0.75 Å, World Precision Instruments). Thus, 100 μl of control vehicle left without larvae (medium only) as a negative control, so that (PEG200:DMSO 1:1; heparin 2 UI/ml) or sample dissolved in vehicle each experimental parameter consisted of 10 to 12 larvae. Results were i.v. infused at a rate of 50 μl/min for 2 min. Ten minutes later, were registered as the average value of the total time of larval move- mice were released from the restrainer and placed in a transparent ment during 30 min.
poly-acrylic cage (32 × 14 × 12.5 cm) for observation. Pentylenetetra-zole was then infused at a rate of 150 μl/min. Seizure manifestation 2.9. EEG recordings stages in mice were scored according to the time between the startof PTZ infusion and the following behavioral events: ear, tail and Zebraﬁsh larvae were allowed to swim in 400 μl of either 10 μg/ml myoclonic twitch, forelimb clonus, falling, tonic hindlimb extension turmeric oil or vehicle for 1 h in a well of a 24-well plate. An equal and death Under these conditions, i.v. infused PTZ triggered volume of 40-mM PTZ was then added to the well. After a 15- all aforementioned behavioral parameters, culminating in death of minute exposure, the larvae were embedded in a 2% low-melting- mice treated with vehicle, approximately 3 min after the start of infu- point agarose. A glass electrode ﬁlled with artiﬁcial cerebrospinal sion. For this reason, behavior was observed up to a maximum of ﬂuid composed of: 124 mM NaCl, 2 mM KCl, 2 mM MgSO4, 2 mM 5 min of PTZ infusion. Any surviving mice were then sacriﬁced. Penty- CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3 and 10 mM glucose (resis- lenetetrazole doses were calculated according to the formula: PTZ tance 1–5 MΩ), was placed into the optic tectum. Recordings were dose [mg/kg] =(PTZ concentration [mg/ml] × infusion rate [ml/ performed in current clamp mode, low-pass ﬁltered at 1 kHz, high-pass s] × infusion duration [s] × 1000)/mouse weight [g].
ﬁltered at 0.1 Hz, digital gain 10, and sampling interval 10 μs (MultiClamp700B ampliﬁer, Digidata 1440A digitizer, both Axon Instruments, USA).
2.11. Statistical analysis The recordings started each time exactly 5 min after removal from theproconvulsant bath and were continued for 10 min.
All statistical analyses were performed using GraphPad Prism 5 A spike was deﬁned as interictal-like if its amplitude exceeded software (GraphPad Software, Inc.). Values were presented as means three times the background and lasted for less than 3 s. Longer ± standard deviation (SD). The locomotor activity of zebraﬁsh larvae discharges were counted as ictal-like ones (Fig. S2). We quantiﬁed was evaluated using one-way ANOVA followed by Dunnett's multiple the number and average duration of either type of electrographic comparison test. Statistically signiﬁcant differences (pb0.05) between activity as well as the total cumulative duration of all forms of a treatment group and the equivalent control groups (vehicle or PTZ) epileptiform discharges during the ten-minute recordings using were considered indicative of decrease or increase in locomotor ac- Clampﬁt 10.2 software.
tivity of zebraﬁsh larvae. For evaluation of the EEG data and the mouseexperiments, signiﬁcant differences (pb 0.05) between estimated time in- 2.10. Generation of PTZ-induced seizures in mice: timed i.v. PTZ infusion tervals prior to above-mentioned seizure stages were calculated using the unpaired Student's t-test.
Mice were randomly divided into groups of ﬁve animals (vehicle and sample). The animals were isolated into individual cages andpre-warmed in front of an infrared lamp for 10 min to dilate the tail 3.1. Evaluation of the anticonvulsant activity of turmeric veins. They were then placed in a restrainer, and the lateral tail veinwas catheterized with a 1-cm long, 29-gauge needle. After conﬁrming The analysis of the methanolic extract of turmeric (C. longa rhi- correct placement, the needle was secured to the tail with surgical zome powder) revealed anticonvulsant activity in the zebraﬁsh larval tape and kept in place until the end of the test. The needle attached PTZ assay (In order to identify the active constituents present to a 0.7-m long polyethylene catheter was connected to two 2.5-ml in the methanolic extract of turmeric, the anticonvulsant properties Fig. 2. HPLC chromatogram of turmeric oil and its major constituents. Peak 4 corresponds to ar-turmerone; peak 5 to α,β-turmerone and peak 6 to α-atlantone.
A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 of curcuminoids and turmeric oil were also assessed through video- tracking analysis (Curcuminoids showed anticonvulsant activ- ity at 2.5 μg/ml (p b0.05) and at 5 and 10 μg/ml (p b 0.01) in our larvalPTZ assay. Further analysis uncovered an additional anticonvulsant activity for turmeric oil. The larvae showed a decrease (p b 0.01) ofPTZ-induced convulsions after exposure to turmeric oil (10 μg/ml).
Notably, exposure of zebraﬁsh larvae to curcuminoids or turmeric oil alone (i.e. in the absence of proconvulsant) also resulted in a slightincrease in locomotor activity compared to vehicle-treated controls(B,C). However, no obvious signs of toxicity (as measured by change in heart rate, loss of posture, lack or delay in response to tac- tile stimuli, or death (see Methods, were observed in these larvae.
3.2. Isolation and evaluation of the anticonvulsant activity of bisabolene sesquiterpenoids from turmeric oil To purify the active constituents in turmeric oil, we performed semi-preparative RP-HPLC analysis. Eight peaks were identiﬁed(and individually collected. From the eight peaks collected,fractions 2 and 7 were not tested in the zebraﬁsh PTZ assay since the collected amounts were not sufﬁcient for carrying out the assay (). Fractions 1, 3 and 8 did not display any anticonvulsant ac-tivity in the zebraﬁsh PTZ assay (data not shown). A signiﬁcant de- crease in the convulsions triggered by PTZ was observed for fractions 4, 5 and 6. Fraction 4 showed anticonvulsant activity at 46 μM (p b 0.05), fraction 5 at 23 μM (pb 0.01) and fraction 6 at con- centrations of 23 μM (p b 0.05) and 46 μM (pb 0.01) Sodiumvalproate, a well-known AED, was included as a positive control for the zebraﬁsh PTZ assay . Sodium valproate showed signiﬁcantactivity at 250 μM (p b0.05) and 500 μM and 1 mM (p b 0.01) (Supple-mentary Fig. S4A).
Fractions 4, 5 and 6 that showed seizure inhibitory activity in the larval zebraﬁsh PTZ assay were further analyzed for chemical structure elucidation. 1H and 13C NMR spectra of fraction 4 are in agreement with reported values for ar-turmerone Nuclearmagnetic resonance analysis indicated fraction 5 as a mixture (1:1)of two isomeric structures identiﬁed as α,β-turmerone by 1D- and 2D-NMR analysis Fraction 6 was identiﬁed as α-atlantone (probably the E-isomer) based on 1H and 13C NMR spectra (data not shown).
Once again, as observed for the curcuminoid and turmeric oil- treated larvae, exposure of zebraﬁsh larvae to α,β-turmerone, ar- Fig. 3. Evaluation of the anticonvulsant activity of bisabolene sesquiterpenoids in thezebraﬁsh PTZ seizure assay. (A) Ar-turmerone; (B) α,β-turmerone and (C) α- turmerone or α-atlantone alone also resulted in a slight increase in atlantone. The x-axis represents the tested concentration for each one of the sesquiter- locomotor activity compared to vehicle-treated controls ().
penoids. The y-axis indicates the total gross locomotor activity exhibited by zebraﬁsh However, no obvious signs of toxicity were observed. This raised the larvae within 30 min. Data are expressed as the mean ± SD (n = 10–12). Statistically question as to whether turmeric oil or its individual constituents signiﬁcant differences between vehicle-treated and sample-treated (white bars) or could perhaps exhibit mild proconvulsant activity.
PTZ-treated and sample plus PTZ-treated groups (gray bars) are labeled as * forp b 0.05 and ** for p b 0.01.
To determine whether turmeric oil can act as a proconvulsant on zebraﬁsh larvae and to conﬁrm whether the decrease in convulsion-like movements observed in larvae co-treated with turmeric oil andPTZ was truly an inhibition of seizure activity, we carried out open-ﬁeld recordings from larval brains. The analysis of electrographic ac- 3.3. Evaluation of the anticonvulsant activity of turmeric, turmeric oil tivity of larvae showed that exposure to 10 μg/ml of turmeric oil and bisabolene sesquiterpenoids in the mouse PTZ-induced seizure model partly protects the larvae from PTZ-induced seizures. Turmeric oilproduced no effect on the number and duration of interictal-like The anticonvulsant activity of curcumin has been described previ- spikes compared to controls ,B,H,I; Supplementary Figs.
ously in mice but no assay for turmeric oil has evaluated its S3C,D) but signiﬁcantly reduced both the number and the duration anticonvulsant properties before. Thus, further conﬁrmation of the of ictal-like discharges (,H,I). Moreover, the latter were ab- positive results obtained in zebraﬁsh with turmeric oil and the bisa- sent in 3 out of ﬁve recordings (Supplementary Fig. S3D). In addition, bolene sesquiterpenoids was performed in the mouse assay. Mice the cumulative duration of all forms of epileptiform discharges treated with turmeric oil (50 mg/kg) showed a signiﬁcant increase quantiﬁed was shortened after turmeric oil exposure E). Im- in PTZ doses required to trigger all behavioral endpoints: forelimb portantly, turmeric oil on its own did not induce epileptiform dis- clonus, falling and tonic hindlimb extension (p b 0.05) and ear, myo- charges in our experimental set-up (F,G; Supplementary clonic, tail twitch, and death (p b0.01), compared to control group Figs. S3A,B).
(). Moreover, a dose of 100-mg/kg turmeric oil in the mouse A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 PTZ assay exhibited signiﬁcant activity in delaying seizure generation for all seizure parameters and death as compared to control (p b 0.01)Regarding the active bisabolene sesquiterpenoids, ar- Turmeric, the powdered rhizomes of C. longa, has been used for turmerone and α,β-turmerone were assessed using the mouse PTZ centuries as a food condiment but also as an ethnomedical treatment seizure model ). Mice infused with a dose of 50 mg/kg of ar- against seizures. We evaluated the anticonvulsant activity of turmeric turmerone exhibited signiﬁcant resistance to the generation of sei- methanolic extract in the zebraﬁsh PTZ-induced seizure model zures leading to an increase in the required dose of PTZ to trigger all with positive results. This ﬁnding is in line with the anticonvulsant assessed events: tonic hindlimb extension (p b 0.05) and ear, myo- properties of turmeric described in previous studies. In these studies, clonic and tail twitch, forelimb clonus, falling and death (p b0.01).
curcumin has often been cited as the principal substance responsible Likewise, the anticonvulsant activity of α,β-turmerone was evaluat- for the activity of turmeric Nevertheless, bioavailability ed, and positive results were also found with a dose of 100 mg/kg analysis of curcumin evidenced poor absorption, rapid metabolism for all seizure parameters: forelimb clonus, falling, ear and tail twitch and excretion impeding its ability to reach the brain in order to (p b 0.05) and myoclonic twitch, tonic hindlimb extension and death exert any potential therapeutic action. Conversely, the other main (p b 0.01). α-Atlantone was not tested in the mouse model since the constituent of turmeric, turmeric oil, has not been evaluated before collected amount was not sufﬁcient to carry out the assay.
for potential anticonvulsant properties.
Sodium valproate was included as positive control in our PTZ The compounds isolated from turmeric oil through semi- tail infusion method for AED screening in mice (Supplementary preparative RP-HPLC were individually evaluated in the zebraﬁsh Fig. S4B). Using this assay, sodium valproate (50 mg/kg) was PTZ assay. This assay revealed signiﬁcant activity for turmeric oil capable of delaying tonic hindlimb extension (p b0.01) and and the major bisabolene sesquiterpenoids: ar-, α,β-turmerone and death (p b 0.05).
α-atlantone. All of the aforementioned constituents exhibited number in 10 min
average duration, s
number in 10 min
average duration, s
cumulative duration, s
Fig. 4. Electrographic evaluation of the anticonvulsant activity of turmeric oil in the larval zebraﬁsh PTZ seizure model. (A–E) Graphical representation of the changes in seizureparameters analyzed. VHC, vehicle-treated, n = 3; TO, turmeric oil-treated, n = 3; VHC + PTZ, vehicle-treated larvae after PTZ exposure, n = 5; TO + PTZ, turmeric oil-treated larvaeafter PTZ exposure, n = 5. (A) Number of interictal-like spikes; (B) average duration of interictal-like spikes; (C) number of ictal-like discharges; (D) average duration of ictal-likedischarges; (E) cumulative duration of all forms of epileptiform activity. (F–I) Representative 2-minute fragments of electrographic activity recordings. (F) Spontaneous electricalactivity in vehicle-treated control; (G) basal activity after exposure to 10-μg/ml turmeric oil; (H) PTZ-induced spiking in vehicle-treated larva; (I) reduced amount of PTZ-inducedepileptiform activity after exposure to 10-μg/ml turmeric oil.
A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 Fig. 4 (continued).
anticonvulsant properties at lower concentrations compared to sodi- The anticonvulsant properties of turmeric oil in the zebraﬁsh um valproate. One possibility for the more potent activity of bisabo- model were successfully corroborated in the mouse PTZ model lene sesquiterpenoids in the zebraﬁsh PTZ assay compared to (50 mg/kg, 100 mg/kg). Further analysis leads to the identiﬁcation sodium valproate could be related to their higher lipophilicity of ar-turmerone and α,β-turmerone as the putative compounds re- (logP). Empirical evidence suggests that small molecules with higher sponsible for the anticonvulsant activity found for turmeric oil in lipophilicity enter zebraﬁsh embryos and larvae more readily than mice. These ﬁndings validate the zebraﬁsh PTZ-induced seizure hydrophilic ones (our own unpublished observations). The bisabo- model as a primary screening tool for identifying novel potential lene sesquiterpenoids display around one hundred times higher lipo- AEDs and reveal the major bisabolene sesquiterpenoids ar-turmerone philicity than sodium valproate. Thus, these results are also in line and α,β-turmerone as anticonvulsant drug candidates to be investigated with the neuroprotection studies in rodent models which have further. At effective doses, neither turmeric oil nor the tested bisabolene shown that turmeric oil and its main bisabolene sesquiterpenoids sesquiterpenoids induced sedation, motor impairment or any other sign easily cross the blood-brain barrier, likely due to their lipophilic na- of toxicity in mice.
ture which allows them to easily pass through cell membranes Interestingly, anticonvulsant properties have been reported also for Turmeric oil and its constituents present better bio- other essential oils such as Laurus nobilis and Cymbopogon winterianus availability and cross biomembranes with less difﬁculty when com- . Although their active constituents have not been isolated to pared to curcumin.
identify the responsible compound/s for the anticonvulsant activity, it
A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 Fig. 6. Evaluation of the anticonvulsant activities of α,β-turmerone and ar-turmeronein the mouse PTZ seizure model. Top panel: table listing PTZ dose/s required to elicitthe indicated seizure behaviors after treatment with bisabolene sesquiterpenoid or ve- Fig. 5. Evaluation of the anticonvulsant activity of turmeric oil in the mouse PTZ seizure hicle only. Graphical depiction of tabulated results from (A) α,β-turmerone at a dose of model. Top panel: table listing PTZ dose/s required to elicit the indicated seizure behaviors 100 mg/kg and (B) ar-turmerone at 50 mg/kg. ‘Control A' column corresponds to after treatment with turmeric oil or vehicle only. Data are expressed as the mean±SD vehicle-treated controls for α,β-turmerone; ‘Control B' column corresponds to (n=5). Graphical depiction of tabulated results from (A) turmeric oil at 50 mg/kg and vehicle-treated controls for ar-turmerone. Data are expressed as the mean ± SD (B) at 100 mg/kg. Results are expressed as relative values compared to control (set as (n = 5). For sake of clarity, SDs are not depicted in the graphs but are indicated in 100%). Statistically signiﬁcant differences between sample (dark gray) and control group the tables. Results are expressed as relative values compared to control (set as 100%).
(light gray) are labeled as * for pb 0.05 and ** for pb 0.01 (unpaired Student's t-test). For Statistically signiﬁcant differences between sample (dark gray) and control group sake of clarity, SDs are not depicted in the graphs but are indicated in the tables. However, (light gray) are labeled as * for p b 0.05 and ** for p b 0.01 (unpaired Student's t-test).
the coefﬁcient of variation never exceeded 28% (unpaired Student's t-test).
For the sake of clarity, SDs are not depicted. However, the coefﬁcient of variationnever exceeded 28% and 37% in the case of ar-turmerone and α,β-turmerone,respectively.
is known that some of their main constituents are monoterpenes, phe-nylpropenes and sesquiterpenes. These compounds present in essentialoils, including the bisabolene sesquiterpenoids from turmeric, show a The identiﬁcation of the pharmacophore/s (functional chemical wide variety of functional groups and differences on their basic carbon group/s) responsible for the anticonvulsant activity of ar-turmerone skeletons. Since the identiﬁed chemical structures of any of the bisabo- and α,β-turmerone will further lead to their target and the probable lene sesquiterpenoids are not similar to the structure of the available mechanisms involved in exerting their activity. It would be of partic- AEDs, this chemical variability may perhaps increase the chances of ular interest if these pharmacophores display their action through a identifying compounds acting through different mechanisms of action.
novel mechanism since this is the major aim of AED discovery. The With regard to the mechanism of action of the bisabolene sesqui- main limitation regarding the discovery of novel AEDs is that most terpenoids, previous studies on the neuroprotective activity of tur- of them have been identiﬁed using the same models, especially MES meric oil have shown that these compounds can suppress oxidative This is probably the main cause for a misguided orientation DNA damage and lipid peroxidation in rodent models . Penty- that has led to the discovery of drugs that act against new-onset epi- lenetetrazole has been shown to increase glutamatergic transmission lepsy but not against the refractory types. Therefore, it is currently in that, in turn, leads to an increase in intracellular calcium and cell our interest to additionally assess the activity of the bisabolene ses- death . Even though the mechanisms of neuroprotection of quiterpenoids in other model/s of epilepsy such as the 6-Hz seizure AEDs have not yet been fully elucidated, several studies have revealed test in mice, the hippocampal kindled rat model of temporal lobe ep- the potential of lamotrigine, levetiracetam, topiramate and zonisa- ilepsy (TLE), etc.
mide to limit DNA damage and restrict the extent of neuronal loss With regard to its toxicity proﬁle, turmeric has been widely used It is, therefore, tempting to speculate that the antioxidant prop- as a food condiment predominantly in India for centuries and is con- erties of the bisabolene sesquiterpenoids are in some way related to sidered safe for human consumption . Furthermore, toxicity stud- their anticonvulsant activities. Clearly, further experiments are war- ies performed in healthy human patients and in silico analysis ranted in order to support this hypothesis.
have predicted ar-turmerone as a safe potential candidate for A.M. Orellana-Paucar et al. / Epilepsy & Behavior 24 (2012) 14–22 further drug development. The absence of toxic effects was also a novel bioenhanced preparation of curcumin. Indian J Pharm Sci 2008;70(4):445–9.
conﬁrmed in our experiments. Larval zebraﬁsh exposed (for up to  Sayyah M, Valizadeh J, Kamalinejad M. Anticonvulsant activity of the leaf essential 24 h) to either turmeric oil or any one of the bisabolene sesquiterpe- oil of Laurus nobilis against pentylenetetrazole- and maximal electroshock- noids displayed normal heart rate and showed no signs of motor induced seizures. Phytomedicine 2002;9(3):212–6.
 Quintans-Júnior LJ, Souza TT, Leite BS, et al. Phytochemical screening and anticon- impairment as evidenced by a normal touch/escape response vulsant activity of Cymbopogon winterianus Jowitt (Poaceae) leaf essential oil in upon application of a tactile stimulus, no balance defects, and no rodents. Phytomedicine 2008;15(8):619–24.
signs of hypoactivity.
 Dohare P, Varma S, Ray M. Curcuma oil modulates the nitric oxide system re- In conclusion, in this study, we demonstrate the usefulness of a sponse to cerebral ischemia/reperfusion injury. Nitric Oxide 2008;19(1):1–11.
 Dohare P, Garg P, Sharma U, Jagannathan NR, Ray M. Neuroprotective efﬁcacy and zebraﬁsh seizure model for rapid bioactivity-guided fractionation of therapeutic window of curcuma oil: in rat embolic stroke model. BMC Comple- natural products and their puriﬁed compounds for the identiﬁcation ment Altern Med 2008;8:55.
of novel small molecules with anticonvulsant activity in vivo. Our  Rathore P, Dohare P, Varma S, Ray A, Sharma U, Jagannathan NR. Curcuma oil: re- duces early accumulation of oxidative product and is anti-apoptogenic in tran- ﬁndings suggest that the main bisabolene sesquiterpenoids of tur- sient focal ischemia in rat brain. Neurochem Res 2008;33(9):1672–82.
meric could be considered as interesting novel AED candidates that  Crawford AD, Esguerra CV, de Witte PA. Fishing for drugs from nature: zebraﬁsh deserve further exploration. Current studies are now underway to as a technology platform for natural product discovery. Planta Med 2008;74(6):624–32.
test these compounds in additional seizure models and to identify  Tan JL, Zon Ll. Chemical screening in zebraﬁsh for novel biological and therapeutic the pharmacophore/s responsible for their anticonvulsant activity discovery. Methods Cell Biol 2011;105:493–516.
through structure–activity relationship (SAR) analysis.
 Lockwood B, Bjerke S, Kobayashi K, Guo S. Acute effects of alcohol on larval zebra- ﬁsh: a genetic system for large-scale screening. Pharmacol Biochem Behav Supplementary materials related to this article can be found on-  Zon L, Peterson R. In vivo drug discovery in the zebraﬁsh. Nat Rev 2005;4(1):35–44.
 Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced changes in zebraﬁsh behavior, neural activity and c-fos expression. Neuroscience  Berghmans S, Hunt J, Roach A, Goldsmith P. Zebraﬁsh offer the potential for a pri- The authors wish to thank R. Ansaloni for her assistance regarding mary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res2007;75(1):18–28.
the microscopic characterization of turmeric used in the present  Winter MJ, Redfern WS, Hayﬁeld AJ, Owen SF, Valentin J-P, Hutchinson TH. Vali- study and N. Cuzco for her work in preparation of turmeric volatile dation of a larval zebraﬁsh locomotor assay for assessing the seizure liability of oil. We are also grateful to I. Smolders and R. Clinckers for sharing early-stage development drugs. J Pharmacol Toxicol Methods 2008;57(3):176–87.
their knowledge and experience with regard to performing the  Mandhane SN, Aavula K, Rajamannar T. Timed pentylenetetrazol infusion test: a mouse PTZ assay and P. Verstreken and R. Habets for providing advice comparative analysis with s.c. PTZ and MES models of anticonvulsant screening and access to their electrophysiology equipment. AMOP was funded in mice. Seizure 2007;16(7):636–44.
 Sills GJ, Butler E, Thompson GG, Brodie MJ. Pharmacodynamic interaction studies through a fellowship from the Vlaamse Interuniversitaire Raad with topiramate in the pentylenetetrazol and maximal electroshock seizure (VLIR)—"Pharmacological Characterization of Medicinal Plants from models. Seizure 2004;13(5):287–95.
the South of Ecuador" Project.
 Jackson B, Snowdon D. Atlas of microscopy of medicinal plants, culinary herbs and spices. London, U.K.: Belhaven Press; 1990. p. 257 p.
 Westerﬁeld M. The zebraﬁsh book. A guide for the laboratory use of zebraﬁsh (Danio rerio). 4th ed. Eugene, Oregon: University of Oregon; 2000.
 He X-G, Lin L-Z, Lian L-Z, Lindenmaier M. Liquid chromatography–electrospray mass spectrometric analysis of curcuminoids and sesquiterpenoids in turmeric  World Health Organization. Atlas: epilepsy care in the world. Geneva, Switzerland: (Curcuma longa). J Chromatogr A 1998;818(1):127–32.
WHO Press; 2005. 90 p.
 Giardina WJ, Gasior M. Acute seizure tests in epilepsy research: electroshock- and  Schmidt D. Drug treatment of epilepsy: options and limitations. Epilepsy Behav chemical-induced convulsions in the mouse. In: Enna SJ, Williams M, Barret JF, Ferkany JW, Kenakin T, Porsolt RD, editors. Current protocols in pharmacology.
 Arroyo S, de la Morena A. Life-threatening adverse events of antiepileptic drugs.
Hoboken, NJ, USA: Wiley & Sons; 2009. p. 27–30.
Epilepsy Res 2001;47(1–2):155–74.
 Scallier A, Massie A, Loyens E, et al. vGLUT2 heterozygous mice show more susceptibil-  Carpay JA, Aldenkamp AP, van Donselaar CA. Complaints associated with the use ity to clonic seizures induced by pentylenetetrazol. Neurochem Int 2009;55(1–3): of antiepileptic drugs: results from a community-based study. Seizure  Kamal A, Shaheer Malik M, Azeeza S, Bajee S, Shaik AA. Total synthesis of (R)- and  Arif H, Buchsbaum R, Weintraub D, Pierro J, Jr. SRR, Hirsch LJ. Patient-reported (S)-turmerone and (7S,9R)-bisacumol by an efﬁcient chemoenzymatic approach.
cognitive side effects of antiepileptic drugs: predictors and comparison of all Tetrahedron Asymmetr 2009;20(11):1267–71.
commonly used antiepileptic drugs. Epilepsy Behav 2009;14(1):202–9.
 Uehara S-I, Yasuda I, Takeya K, Itokawa H. New bisabolane sesquiterpenoids from  Meador KJ. Cognitive and memory effects of the new antiepileptic drugs. Epilepsy the rhizomes of Curcuma xanthorrhiza (Zingiberaceae). Chem Pharm Bull  Schachter SC. Botanicals and herbs: a traditional approach to treating epilepsy.
 Friesen RW, Blouin M. Preparation of γ,δ‐unsaturated β‐ketophosphonates from tertiary α‐allenic alcohols. The synthesis of (+/−)‐(E)‐α‐atlantone. J Org Chem  Nsour W, Lau CB-S, Wong IC. Review on phytotherapy in epilepsy. Seizure  Bharal N, Sahaya K, Jain S, Mediratta PK, Sharma KK. Curcumin has anticonvulsant  Malawska B. New anticonvulsant agents. Curr Top Med Chem 2005;5(1):69–85.
activity on increasing current electroshock seizures in mice. Phytother Res  Willmore LJ, Losigamone, Dr Willmar Schwabe. Curr Opin Investig Drugs  Ribeiro MCP, de Ãvila DS, Schneider CYM, et al. alpha-Tocopherol protects against  World Health Organization. WHO monographs on selected medicinal plants.
pentylenetetrazol- and methylmalonate-induced convulsions. Epilepsy Res Geneva, Switzerland: WHO library cataloguing in publication data; 1999. 289 p.
 Jyoti A, Sethi P, Sharma D. Curcumin protects against electrobehavioral progres-  Lores Arnaiz S, Travacio M, Llesuy S, Rodríguez de Lores Arnaiz G. Regional vulner- sion of seizures in the iron-induced experimental model of epileptogenesis.
ability to oxidative stress in a model of experimental epilepsy. Neurochem Res Epilepsy Behav 2009;14(2):300–8.
 Jithendra C, Murthy TE, Upadyay L. Protective role of curcumin in maximal elec-  Willmore LJ. Antiepileptic drugs and neuroprotection: current status and future troshock induced seizures, memory impairment and neurotransmitters in rat roles. Epilepsy Behav 2005;7(Suppl. 3):S25–8.
brain. JPCCR 2008;2(1):35–9.
 Löscher W, Schmidt D. Modern antiepileptic drug development has failed to de-  Sumanont Y, Murakami Y, Tohda M, Vajragupta O, Watanabe H, Matsumoto K.
liver: ways out of the current dilemma. Epilepsia 2011;52(4):657–78.
Prevention of kainic acid-induced changes in nitric oxide level and neuronal cell  Villegas I, Sánchez-Fidalgo S, Alarcón de la Lastra C. New mechanisms and thera- damage in the rat hippocampus by manganese complexes of curcumin and diace- peutic potential of curcumin for colorectal cancer. Mol Nutr Food Res 2008;52: tylcurcumin. Life Sci 2006;78(16):1884–91.
 Mehla J, Reeta KH, Gupta P, Gupta YK. Protective effect of curcumin against sei-  Joshi J, Ghaisas S, Vaidya A, et al. Early human safety study of turmeric oil zures and cognitive impairment in a pentylenetetrazole-kindled epileptic rat (Curcuma longa oil) administered orally in healthy volunteers. J Assoc Physicians model. Life Sci 2010;87(19–22):596–603.
 Antony B, Merina B, Iyer VS, Judy N, Lennertz K, Joyal S. A pilot cross-over  Balaji S, Chempakam B. Toxicity prediction of compounds from turmeric study to evaluate human oral bioavailability of BCM-95®CG (Biocurcumax™), (Curcuma longa L). Food Chem Toxicol 2010;48(10):2951–9.
Phillip A. Glogoza, Extension EntomologistDean K. McBride, Professor EmeritusAlbin W. Anderson, Professor Emeritus North Dakota State UniversityFargo, North Dakota 58105 At least 43 species of mosquitoes are known tooccur in North Dakota. Fortunately, only a few species cause annoyance. Nevertheless, their presence affects people engaged in outdoor activities during the warm months of the year.