Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Lavender Oil-Potent Anxiolytic Properties via Modulating Voltage Dependent Calcium Channels

Abstract

Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.

Introduction

Lavender oil (LA) is an important part of our today's aromatherapy to promote “well-being” and to reduce distress and “ill-being”. “Well-being” is a psychological construct comprising several domains related to personality including self- acceptance and purpose in life among others [1]. Thus, changes induced by LA might be more directed to improvement of ill-being and distress which show overlap with anxiety and tension at the biological level [2]. When applied by inhalation LA has been associated not only with feelings of pleasantness but also with some improving effects on mood and anxiety [3], [4]. While most if not all of those effects of lavender oil in aromatherapy may be mediated by its pleasant odour there is increasing evidence strongly suggesting a pharmacodynamic effect of LA independent of its odour when applied systemically. I) Anxiolytic properties have been demonstrated for LA in experimental animals following inhalation of very high concentrations but also after i.p. or oral administration [5][8]. II) When given in capsules containing 100 or 200 µl LA, anxiolytic properties have been shown in human volunteers following stressful situations [9]. III) Recent clinical trials using Silexan, a standardized LA oil preparation, showed pronounced effects in patients with subsyndromal or subthreshold anxiety disorders as well as in patients with Generalized Anxiety Disorder (GAD) after oral administration. Importantly, Silexan was similarly active compared to the benzodiazepine lorazepam (0,5 mg) during 6 weeks of treatment [10] in patients suffering from GAD. Silexan is a patented active substance produced from Lavandula angustifolia flowers by steam distillation consisting of the main active constituents. linalool (36.8%) and linalyl acetate (34.2%). Silexan (active substance of Lasea®, available as immediate release soft gelatine capules containing 80 mg) has been licensed in Germany for the oral treatment of subsyndromal anxiety and tension in 2009.

Even if several preclinical behavioural pharmacological studies and the new clinical data clearly show the anxiolytic activity of LA and especially of Silexan, the molecular mechanism of action explaining these positive effects was missing. In contrast to previous studies, we used physiological relevant concentrations of Silexan which where found in pharmacokinetic experiments. First, we showed anxiolytic effects of Silexan at these low concentrations in behavioural pharmacological tests such as the elevated plus maze. Second, Silexan showed similar effects compared to the established anxiolytics diazepam and pregabalin. To decipher the molecular mechanism of Silexan, we tested whether Silexan modulates the activity of voltage operated calcium channels (VOCCs) since Silexan did not reveal any affinity to known targets of other anxiolytic drugs (SERT, NET, DAT, MAO-A and the GABAA-receptor; data not shown). Under pathological conditions like anxiety or stress disorders, it has been speculated that enhanced Ca2+-influx mainly through N and P/Q type VOCCs may increase the release of neurotransmitters such as glutamate and norepinephrine [11], [12] which are involved in the pathogenesis of these diseases. Third, we show for the first time that Silexan unselectively inhibits several VOCCs, such as P/Q-type and N-type VOCCs using a broad set of methods including calcium imaging as well as patch clamp technique. In addition, we were able to demonstrate that Silexan does not bind to the binding site of pregabalin at the P/Q type calcium channels. Pregabalin modulates P/Q type VOCCs after binding at the auxiliary α2δ-1 or -2 subunits and thereby reduces Ca2+-influx through these channels [13], [14]. Taken together, we elucidate the anxiolytic mechanism of action of LA and thereby provide a molecular rationale for the clinical use of Silexan.

Materials and Methods

Animals

Female 2–3 months old NMRI mice with an average weight of 30 g were used for the preparation of synaptosomes, purified synaptosomal membranes and for the determination of pentobarbital sleep time. Male 2–3 months old NMRI mice were utilized for the elevated plus maze test. For the preparation of primary hippocampal neurons female time-mated Sprague-Dawley rats were used. Animals were purchased from Charles River Laboratories, Sulzfeld, Germany or Janvier SAS, St. Berthevin, France. All animal care and experimental procedures were in concordance with the German law on animal care and handling of animals and the guidance of the European Community Council Directive, and the protocol was approved by the local commission for the Care and Use of Laboratory Animals of the Government by Baden-Württemberg (Regierungspräsidium Karlsruhe, permit numbers 35-9185.82/A-33/04 and 35-9185.82/A-31/04). All animals were housed in plastic cages with water and food ad libitum and were maintained on a 12 h light/dark cycle.

Drugs

Silexan, linalool, linalyl acetate, diazepam and pregabalin were kindly provided by Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany. Pentobarbital sodium salt, ω-agatoxin IVA, nifedipine, arachidonylethanolamide, pertussis toxin, geraniol, 1,8-cineole and fura-2-AM were purchased from Sigma-Aldrich, Taufkirchen, Germany. ω-conotoxin GVIA was obtained from Tocris Bioscience, Bristol, UK and dihydrolinalool from TCI Europe, Eschborn, Germany. [3H]-gabapentin was obtained from Biotrend, Cologne, Germany.

Elevated Plus Maze test

Male NMRI mice (8 per group) were treated orally for 3 days with doses between 1 and 30 mg/kg/day Silexan, 0.2% agar suspension (10 ml/kg), diazepam (2.5 mg/kg) or pregabalin (100 mg/kg) as positive controls. Anxiety-related behaviour was tested 1 h after the last treatment in a standard elevated plus maze apparatus (central platform 5×5 cm, open arms 30×5 cm, closed arms 30×5×15 cm), 60 cm above the floor. The number of entries and the time spent in the open arms was recorded during 5 minutes.

Pentobarbital sleep time

Female NMRI mice were treated orally for 9 days with dosages between 1 and 30 mg/kg Silexan, 0.2% agar suspension (10 ml/kg) or pregabalin (100 mg/kg). Pentobarbital (45 mg/kg) was diluted in physiological saline and administered i.p. to each mouse one hour after last treatment. The sleeping time was defined as the lapse of time required to change from dorsal position to the normal position.

Isolation and plating of rat P0–P2 hippocampal neurons

Primary hippocampal neurons were prepared from P0–P2 Sprague-Dawley rat pups according to Amaral et al using the Worthington Papain Dissociation Kit (Worthington, Lakewood, NJ) [15]. Cells were plated on poly-D-lysine/laminin coated glass cover slips in serum free Neurobasal A media containing L-glutamine (1 mM) and 2% B27 in 6 well plates at a density of 2×105 cells per well. Neurons were grown in 37°C in a humidified incubator containing 5% CO2 in air. Cultures were maintained for 14 days before experimental procedures.

Calcium measurements in primary hippocampal neurons

Intracellular Ca2+ measurements were conducted according to literature [16], [17]. After 14 days, hippocampal neurons on cover slips were loaded with 1 µM fura-2-AM for 30 min and then placed on the stage of an inverted Axiovert S100 microscope (Zeiss, Oberkirchen, Germany). Intracellular calcium concentrations were measured by dual excitation (340/380 nm) ratio method.

Synaptosomal preparations for intracellular calcium measurements

Murine synaptosomes from whole brain without cerebellum were prepared as described previously [18]. Synaptosomal samples were loaded with 5 µM fura-2-AM for 40 min. Fura-2-signals were calibrated according to the method of Grynkiewicz et al, using a KD value of 224 nM [19]. For the determination of Rmax and Rmin 0,2% sodium dodecyl sulphate and tris(hydroxymethyl)aminomethane (Tris) 30 mM/ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid (EGTA) 6 mM, respectively were added.

[3H]-gabapentin binding studies

Partially purified synaptic membranes were prepared from mouse cortex using sucrose density gradients according to Suman-Chauhan et al [20]. Protein content of synaptic membrane suspension was determined by the Lowry protein assay [21]. For [3H]-gabapentin binding studies, tissue (0.1 mg protein) was incubated with 20 nM [3H]-gabapentin in 10 mM HEPES buffer (pH 7.4 at RT) in the presence of varying concentrations of test compound for 40 min. Afterwards, the samples were filtered through Whatman GF/B filters under vacuum. Filters were washed 3 times with 5 ml ice-cold 100 mM NaCl. Bound radioactivity was determined using liquid scintillation counting. Non-specific binding was defined by 1 mM pregabalin.

Electrophysiological recordings

For measurement of N- and P/Q-type currents, different transfected cell lines were applied, each expressing only one VOCC subtype. N-type channel recordings were carried out on CHO cells stably expressing Cav2.2, α2δ-1 and β-subunits. The cell line was kindly provided by B. Fakler, Institute of Physiology II, University of Freiburg, Germany. Cells were grown in Minimal Essential Medium (MEM) ALPHA including 10% fetal calf serum, L-glutamine 200 mM, G418 0.7 mg/ml, hygromycin B 0.25 mg/ml and blasticidin 5 µg/ml. For P/Q-type current analysis, HEK 293 cells were transiently transfected with α2δ-1 and β3a subunits, as previously described, whereas the α1A subunit was cloned in a pEGFP-N1 vector [22].

Whole cell calcium currents were recorded using the HEKA-10 patch clamp amplifier (HEKA Electronic, Lambrecht, Germany). For measurement of calcium current inhibition by Silexan, extracellular solution containing vehicle or Silexan was applied by superfusion and currents were recorded at 1 Hz during a 50 ms depolarizing pulse to 20 mV. The extracellular solution contained (in mM): CaCl2 10, tetraethylammonium chloride (TEA-Cl) 125, HEPES 10 and glucose 5. The pH was adjusted to 7.4 using TEA-OH. The intracellular solution contained (in mM): CsCl 110, HEPES 10, EGTA 10, MgCl2 3, Na-adenosine triphosphate (Na-ATP) 3 and guanosine 5-triphosphate tris salt (GTP-Tris) 0.6. The pH was adjusted to 7.2 using CsOH. Patch pipettes were pulled from borosilicate glass tubing (Sutter Instruments, Novato, CA, USA) with input resistance ranged between 1.8 and 4.0 MΩ. Cell capacitance ranged between 12 and 25 pF for N-type cells and 10 and 50 pF for P/Q-type cells. Series resistance was typically between 2.5 and 8 MΩ and was compensated up to 70%. Data were recorded with HEKA pulse 8.5 software package and analysed off-line. The holding potential in experiments with N- or P/Q-type cells was −80 mV.

Statistical analysis

Results were expressed as mean ± SEM. Statistical analyses were performed with two-sided t-tests. Stars represent p-values: ***p<0.001, **p<0.01, *p<0.05.

Results

Silexan improves anxiolytic behaviour in the elevated plus maze test

Several groups previously showed anxiolytic effects of LA preparations given by inhalation not standardized concerning their composition [6], [7]. None of these studies dealt with anxiolytic effects of oral administered LA in dosages which are in accordance with the dosage of 80 mg/d used in humans. Therefore, we tested the anxiolytic activity of Silexan in the elevated plus maze test, a well established anxiety model. Mice were treated orally for 3 days with Silexan, the benzodiazepine diazepam or the gabapentinoid pregabalin as active controls. As expected, diazepam and pregabalin both increased the time spent in the open arm (Fig. 1B and C) as well as the number of entries into the open arm. Already at a concentration of 1 mg/kg BW Silexan showed a significant elevation of the number of entries into the open arm (Fig. 1A). This anxiolytic effect is dose-dependent and already maximal at a concentration of 3 mg/kg BW. Similar to diazepam and pregabalin, Silexan also elevated the pentobarbital sleeping time (Fig. 1D) at similar concentrations (3–30 mg/kg) in accordance with Linck et al [8]. As Silexan does not show sedation in humans, this finding rather suggests sleep promoting than sedative properties [9], [10], [23][26].

thumbnail
Figure 1. Silexan reduces anxiety-related behaviour in the elevated plus maze and increases pentobarbital-induced sleeping time.

(A–C) Mice were treated orally with diazepam (2.5 mg/kg BW), pregabalin (100 mg/kg BW) or Silexan (1–30 mg/kg BW) for three days. Diazepam, pregabalin and Silexan increased the time spent in the open arm (A, B) and entry number (C). (D) Mice were treated orally with pregabalin (100 mg/kg BW) or Silexan (1–30 mg/kg BW) for nine days. Sleeping time was determined after i.p. application of pentobarbital (45 mg/kg BW). All data presented are mean values ± SEM using 8 mice per treatment group (unpaired t-test).

https://doi.org/10.1371/journal.pone.0059998.g001

Similar effects of Silexan and pregabalin on VOCCs in murine synaptosomes

Silexan showed no affinity to known targets of other anxiolytic drugs such as SERT, NET, DAT, MAO-A and the GABAA-receptor (data not shown). Therefore, we investigated if Silexan shares its mode of action with the gabapentinoid pregabalin which targets the α2δ-1 and -2 subunits of VOCCs. We examined, if Silexan and pregabalin inhibit Ca2+-influx in murine synaptosomes (Fig. 2A–C). VOCCs were activated by KCl. Under these conditions pregabalin inhibited Ca2+-influx substantially (Fig. 2A, 2C). Maximal effects were already seen at a concentration of 3 µM reducing the Ca2+-influx to 65.86±6.71%. Increasing pregabalin concentrations did not show any additional effect. Silexan dose-dependently reduced KCl-induced Ca2+-increase in a biphasic mode, showing an inhibition of about 20% at concentrations between 0.1 and 1 µg/ml(Fig. 2B, 2C). Contrary to pregabalin, Silexan further decreased Ca2+-elevation at higher concentrations, exceeding pharmacologically relevant plasma levels in man and animal by several orders of magnitude (data not shown). The major ingredients of Silexan are linalool and linalyl acetate which each make up to about 35% of the product. However, linalool and linalyl acetate are similarly active when applied directly to the synaptosomes (Fig. 2E). Due to the fact that linalyl acetate is completely metabolized to linalool, we estimated an IC50 value for linalool of about 37 nM (Fig. 2D).

thumbnail
Figure 2. Silexan dose-dependently inhibits VOCCs in murine synaptosomes.

Dose-dependent effects of pregabalin (A), Silexan (B) and linalool (D) on KCl-induced Ca2+-influx. Murine synaptosomes were preincubated (10 min) with different concentrations of pregabalin (A, 0.03–100 µM), Silexan (B, 0.01–10 µg/ml) or linalool (D, 0.01–1 µM) and afterwards stimulated with KCl (80 mM). (C) Representative Ca2+-transients in murine synaptosomes after KCl-induced activation of VOCCs in the presence and absence of pregabalin (3 µM) and Silexan (1 µg/ml). (E) Effect of the preincubation (10 min) with linalool (1 µM) and linalyl acetate (1 µM) on KCl-induced Ca2+-influx in murine synaptosomes. (F) Several other monterpenes have no effect on KCl-induced Ca2+-influx in murine synaptosomes under similar conditions. All data presented are mean values ± SEM (n = 10–12), paired t-test.

https://doi.org/10.1371/journal.pone.0059998.g002

One possible explanation for this effect could be a nonspecific modulation of channels properties by alterations of membrane fluidity. However, using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene and mouse brain membranes according to Kirsch et al. [27], no effect of Silexan on membrane fluidity was seem up to concentrations of 100 µg/ml.

To further investigate if the Silexan and linalool mediated effects on VOCCs are selective, we additionally investigated several terpenes with closely related chemical structures such as geraniol, cineol and dihydrolinalool. Importantly, none of the other terpenes inhibited KCl induced calcium influx (Fig. 2F) supporting the idea that this effect is specific for linalool and lavender oil and is not a common phenomenon of lipophilic monoterpenes.

Silexan also inhibits VOCCs in primary hippocampal neurons

To further characterize the effects of Silexan on VOCCs, we decided to study KCl dependent Ca2+-influx in primary hippocampal neurons because of the important role of the hippocampus for regulating emotions and anxiety. Similar to the effects obtained in synaptosomes, Silexan inhibited significantly and dose-dependently KCl induced Ca2+-increase (Fig. 3A, C, D, G) in concentrations between 0.1 and 30 µg/ml. In this model, pregabalin decreased Ca2+ currents to 62.84±8.64% of ctl (30 µM; Fig.3B, C, E, H).

thumbnail
Figure 3. Silexan and pregabalin show similar inhibitory effects on VOCCs in primary hippocampal neurons.

Concentration-dependent effect of Silexan (A, 0.01–10 µM) and pregabalin (3–30 µM) after 10 min preincubation on KCl (60 mM) induced Ca2+-influx in primary hippocampal neurons. (C) shows representative traces of intracellular Ca2+ concentration in primary hippocampal neurons stimulated with KCl (60 mM) preincubated with Silexan (1 µg/ml), pregabalin (10 µM) compared to ctl. (D)–(F) show representative fields of fura-2 loaded hippocampal neurons preincubated with Silexan 1 µg/ml (D), pregabalin 10 µM (E) or control (F) under basal conditions. (G)–(I) show the same samples after stimulation with KCl (60 mM). All data presented are means ± SEM (n = 10–14, paired t-test).

https://doi.org/10.1371/journal.pone.0059998.g003

Silexan does not bind to the gabapentin binding site at α2δ-subunits of P/Q-type VOCCs

The specific effects of pregabalin and the structurally related drug gabapentin on presynaptic VOCCs of the P/Q-type have been associated with the specific binding to the modulating and partially extracellularly located subunit α2δ-1 and -2, which can be labelled by [3H]-gabapentin binding [13], [28]. In agreement with the literature pregabalin inhibited specific [3H]-gabapentin binding with an IC50 value of about 70 nM (Fig. 4A) [29]. In contrast, no effect on [3H]-gabapentin binding was observed for Silexan (Fig. 4B). Thus, even if both drugs seem to interact with VOCCs, they probably do not share the same binding site at VOCCs.

thumbnail
Figure 4. Silexan does neither share the binding site of pregabalin, nor inhibits VOCCs via Gi-coupled receptors.

(A) Displacement studies conducted with [3H]-gabapentin in partially purified synaptic membranes. Synaptic membranes were incubated with [3H]-gabapentin in presence of pregabalin (A, 0.001–100 µM) or Silexan (B, 0.1–100 µM; n = 3). (C) Primary hippocampal neurons were incubated in the presence or absence of PTX (200 ng/ml) for 18 h. Afterwards cells were treated with Silexan (1 µg/ml) or AEA (1 µM) for 10 min and then stimulated with KCl (60 mM; n = 11–12). (D) Additive inhibitory effects of P/Q-type and N-type channel blockers on KCl-induced Ca2+-influx in murine synaptosomes. Synaptosomes were preincubated with ω-agatoxin IVA (100 nM), ω-conotoxin GVIA (30 nM) or a combination of both inhibitors for 10 min. Afterwards, they were stimulated with KCl (80 mM). Silexan (1 µg/ml) causes no significant additive effects when combined with the N-type VOCCs inhibitor ω-conotoxin GVIA (30 nM, E), or the P/Q-type inhibitor ω-agatoxin IVA (100 nM, F; n = 9–12). All data presented are mean values ± SEM (paired t-test).

https://doi.org/10.1371/journal.pone.0059998.g004

Gi coupled G-protein receptors are not involved in VOCCs inhibition by Silexan

Modulation of VOCCs could either occur via direct action on the VOCC protein complex or indirect via activation of upstream Gi coupled receptors, e.g. cannabinoid receptors [30]. We measured KCl-induced Ca2+-elevation in primary hippocampal neurons treated with Silexan in the presence or absence of pertussis toxin (PTX). PTX specifically and irreversibly inactivates Gi protein coupled receptors via catalysis of Gαi subunit ADP-ribosylation [31]. Silexan reduced significantly depolarisation induced Ca2+- influx independent of PTX treatment (Fig. 4C). Arachidonylethanolamide was used as positive control. The endogenous ligand for the CB1 cannabinoid receptor decreased KCl-induced Ca2+-influx to 44.26±9.37% [32]. Due to Gi protein involvement, the inhibitory effect was almost completely abolished after PTX treatment (91.28±16.22%). Taken together, Silexan does not interact with Gi coupled G-protein coupled receptors.

Silexan inhibits different classes of VOCCs

To elucidate if Silexan preferentially inhibits P/Q type VOCCs such as pregabalin, we deternined the effects of Silexan in the presence of the P/Q type inhibitor ω-agatoxin IVA and the N-type inhibitor ω-conotoxin GVIA because these two channel types mainly promote the KCl mediated Ca2+-increase in primary hippocampal and in synaptosomes (please also see Supplementary Figure S1).

Both toxins used in this study target ligand-binding pockets at the α1 subunit and have been reported to act additively [33], [34]. This was confirmed in our experiments. After single toxin incubation, ω-agatoxin IVA reduced KCl-induced Ca2+-elevation to 65.02±8.88%, while ω-conotoxin GVIA diminished Ca2+-currents to 68.44±3.16%. Joint application of the two toxins lowered KCl-evoked increase in intrasynaptosomal Ca2+-concentrations to 40.43±3.55% (Fig. 4D). To test if Silexan acts specifically at a single VOCC subtype, we performed combined application measurements with ω-conotoxin GVIA (30 nM; Fig. 4E) or ω-agatoxin IVA (100 nM; Fig. 4F). When both toxins were applied together with Silexan, no relevant additive effects were observed. This observation strongly argues against a specific effect of Silexan at one of the individual VOCCs.

This last observation prompted us to further investigate the effects of Silexan on the properties of VOCCs using the patch-clamp technique. In CHO cells stably expressing N-type VOCC, Silexan displayed a concentration-dependent inhibition of Ca2+ currents (Fig. 5A/C/D). Silexan 1 µg/ml already reduced Ca2+ currents significantly by about 10%. At the higher concentration of 10 µg/ml Ca2+ currents were additionally inhibited to about 80%. The currents recover after washing out Silexan 1 µg/ml and 10 µg/ml (Fig. 5 E,F). Silexan also affected other VOCCs. This could be demonstrated in transiently transfected HEK 293 cells, expressing the P/Q-type channel (Fig. 5B). Silexan caused a reduction of Ca2+ currents in P/Q-type expressing cells to a similar extent and in a similar dose-dependent fashion. Electrophysiological recordings in HEK 293 cells stably expressing the T-type VOCC revealed that Silexan also inhibits low voltage activated currents at this channel (data not shown). Taken together, our findings suggest that Silexan acts non-specifically at different classes of VOCC.

thumbnail
Figure 5. Silexan inhibits N- and P/Q-type VOCCs in whole cell patch-clamp experiments.

(A) Whole cell recordings of N-type VOCCs were conducted in CHO cells stably expressing N-type Ca2+ channels. Silexan (1 or 10 µg/ml) was applied by superfusion for 50 s. The cells were stimulated with a depolarizing pulse to 20 mV at 1 Hz and the amplitude was recorded (n = 9–14). (C, D) Representative currents of whole cell patch clamp recordings with Silexan 1 and 10 µg/ml in N-type cells when stimulated with a depolarizing pulse to 20 mV before (ctl) and at the end of Silexan application (+ Silexan). (B) Effect of preincubation of Silexan (1–10 µM) on depolarizing pulses to 20 mV in P/Q-type cells (n = 4–5). (E and F) Time course of the N-type Ca2+ channel peak current amplitude for 125 seconds by depolarizing pulses to +20 mV at a pulse frequency of 1 Hz. Inhibition of the N-type Ca2+ channel peak current amplitude by Silexan 1 µg/ml (E) or 10 µg/ml (F) compared to DMSO. The arrows indicate the time points of the Silexan application and the washout. A total number of 10 (DMSO), 14 (Silexan 1 µg/ml) and 9 cells (Silexan 10 µg/ml) were averaged. All data presented are mean values ± SEM (unpaired t-test).

https://doi.org/10.1371/journal.pone.0059998.g005

Discussion

In the past, several mechanisms for lavender oil's beneficial effects on anxiety and mood were postulated, including interactions with NMDA or GABAA receptors, voltage dependent sodium channels, or glutamatergic and cholinergic neurotransmission [35][39]. Most of these effects were observed in the millimolar or high micromolar concentration range.

Here we show three novel findings. First, Silexan shows anxiolytic effects in the elevated plus maze test at low oral doses (1–10 mg/kg BW) corresponding to dosages given in humans (80 mg/d). Second, our studies revealed an inhibition of VOCCs by Silexan at nanomolar concentrations. Third, pregabalin and Silexan do not share the molecular target, the α2δ subunit of P/Q type channels [13], [40]. In addition, Silexan did not inhibit Ca2+-increase via VOCCs as a consequence of previous activation of Gi coupled receptors. Silexan unselectively modulates with similar potency several VOCCs such as the N-type and the P/Q type VOCCs. This parallels to some extent pregabalin, which acts on the VOCC channel complex via the α2δ subunit but mainly inhibits P/Q type calcium channels. The precise mechanisms of the channel modulation by Silexan are not yet known but may be either direct by binding and inhibiting the channel complex or more indirect by influencing the properties or surface expression of VOCC subunits.

Taken together, we shed light into the molecular mechanism of action of LA in anxiety and thereby might help to further establish the clinical use of Silexan in patients suffering from anxiety.

Supporting Information

Figure S1.

Similar expression pattern of VOCCs in murine synaptosomes and primary hippocampal neurons. μ Murine synaptosomes (A–C) and primary hippocampal neurons (D–F) were preincubated with the selective P/Q type blocker ω-agatoxin IVA (A/D), the selective N type blocker ω-conotoxin GVIA (B/E) and the L type blockers verapamil and nifedipine (C/F) and afterwards stimulated with KCl. All data represent mean ± SEM (n = 8–12; paired t-test). (G)–(J) Representative confocal images of primary hippocampal neurons stained with a specific primary antibody against P/Q-type VOCCs (G), N-type VOCCs (H), and L-type VOCCs (I,J).

https://doi.org/10.1371/journal.pone.0059998.s001

(TIF)

Author Contributions

Conceived and designed the experiments: AMS MN TW NK KL WEM. Performed the experiments: AMS MN TW. Analyzed the data: AMS NM TW NK KL WEM. Wrote the paper: AMS KL WEM.

References

  1. 1. Ryff CD, Singer B (1996) Psychological well-being: Meaning, measurement, and implications for psychotherapy research. Psychother Psychosom 65: 14–23.
  2. 2. Ryff CD, Singer BH (2008) Know thyself and become what you are: A eudaimonic approach to psychological well-being. J Happiness Stud 9: 13–39.
  3. 3. Moss M, Cook J, Wesnes K, Duckett P (2003) Aromas of rosemary and lavender essential oils differentially affect cognition and mood in healthy adults. Int J Neurosci 113: 15–38.
  4. 4. Lehrner J, Marwinski G, Lehr S, Johren P, Deecke L (2005) Ambient odors of orange and lavender reduce anxiety and improve mood in a dental office. Physiol Behav 86: 92–95.
  5. 5. Umezu T, Nagano K, Ito H, Kosakai K, Sakaniwa M, et al. (2006) Anticonflict effects of lavender oil and identification of its active constituents. Pharmacol Biochem Behav 85: 713–721.
  6. 6. Shaw D, Annett JM, Doherty B, Leslle JC (2007) Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats. Phytomedicine 14: 613–620.
  7. 7. Bradley BF, Starkey NJ, Brown SL, Lea RW (2007) Anxiolytic effects of Lavandula angustifolia odour on the Mongolian gerbil elevated plus maze. J Ethnopharmacol 111: 517–525.
  8. 8. Linck VD, da Silva AL, Figueiro M, Piato AL, Herrmann AP, et al. (2009) Inhaled linalool-induced sedation in mice. Phytomedicine 16: 303–307.
  9. 9. Bradley BF, Brown SL, Chu S, Lea RW (2009) Effects of orally administered lavender essential oil on responses to anxiety-provoking film clips. Hum Psychopharmacol 24: 319–330.
  10. 10. Woelk H, Schlafke S (2010) A multi-center, double-blind, randomised study of the Lavender oil preparation Silexan in comparison to Lorazepam for generalized anxiety disorder. Phytomedicine 17: 94–99.
  11. 11. Musazzi L, Racagni G, Popoli M (2011) Stress, glucocorticoids and glutamate release: Effects of antidepressant drugs. Neurochem Int 59: 138–149.
  12. 12. Kalk NJ, Nutt DJ, Lingford-Hughes AR (2011) The role of central noradrenergic dysregulation in anxiety disorders: evidence from clinical studies. J Psychopharmacol 25: 3–16.
  13. 13. Dooley DJ, Donovan CM, Meder WP, Whetzel SZ (2002) Preferential action of gabapentin and pregabalin at P/Q-type voltage-sensitive calcium channels: Inhibition of K+-evoked [H-3]-norepinephrine release from rat neocortical slices. Synapse 45: 171–190.
  14. 14. Fink K, Dooley DJ, Meder WP, Suman-Chauhan N, Duffy S, et al. (2002) Inhibition of neuronal Ca2+ influx by gabapentin and pregabalin in the human neocortex. Neuropharmacology 42: 229–236.
  15. 15. Amaral MD, Pozzo-Miller L (2007) TRPC3 channels are necessary for brain-derived neurotrophic factor to activate a nonselective cationic current and to induce dendritic spine formation. J Neurosci 27: 5179–5189.
  16. 16. Treiber K, Singer A, Henke B, Muller WE (2005) Hyperforin activates nonselective cation channels (NSCCs). Br J Pharmacol 145: 75–83.
  17. 17. Leuner K, Li W, Amaral MD, Rudolph S, Calfa G, et al. (2013) Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca(2+)-permeable TRPC6 channels. Hippocampus 23: 40–52.
  18. 18. Leuner K, Heiser JH, Derksen S, Mladenov MI, Fehske CJ, et al. (2010) Simple 2,4-Diacylphloroglucinols as Classic Transient Receptor Potential-6 Activators-Identification of a Novel Pharmacophore. Mol Pharmacol 77: 368–377.
  19. 19. Grynkiewicz G, Poenie M, Tsien RY (1985) A New Generation of Ca-2+ Indicators with Greatly Improved Fluorescence Properties. J Biol Chem 260: 3440–3450.
  20. 20. Sumanchauhan N, Webdale L, Hill DR, Woodruff GN (1993) Characterization of [3H]Gabapentin Binding to A Novel Site in Rat-Brain-Homogenate Binding-Studies. Eur J Pharmacol 244: 293–301.
  21. 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein Measurement with the Folin Phenol Reagent. J Biol Chem 193: 265–275.
  22. 22. Klugbauer N, Dai SP, Specht V, Lacinova L, Marais E, et al. (2000) A family of gamma-like calcium channel subunits. FEBS Lett 470: 189–197.
  23. 23. Kasper S (2010) Silexan, an orally administered Lavandula oil preparation, is effective in the treatment of anxiety disorders and related conditions. Planta Med 76: 1184–1184.
  24. 24. Kasper S, Gastpar M, Mueller WE, Volz HP, Moeller HJ, et al. (2010) Efficacy and safety of silexan, a new, orally administered lavender oil preparation, in subthreshold anxiety disorder-evidence from clinical trials. Wien Med Wochenschr 160: 547–556.
  25. 25. Kasper S, Gastpar M, Muller WE, Volz HP, Moller HJ, et al. (2010) Silexan, an orally administered Lavandula oil preparation, is effective in the treatment of 'subsyndromal' anxiety disorder: a randomized, double-blind, placebo controlled trial. Int Clin Psychopharmacol 25: 277–287.
  26. 26. Uehleke B, Schaper S, Dienel A, Schlafke S, Stange R (2012) Phase II trial on the effects of Silexan in patients with neurasthenia, post-traumatic stress disorder or somatization disorder. Phytomedicine 19: 665–671.
  27. 27. Kirsch C, Eckert GP, Mueller WE (2003) Statin effects on cholesterol micro-domains in brain plasma membranes. Biochem Pharmacol 65: 843–856.
  28. 28. Dooley DJ, Taylor CP, Donevan S, Feltner D (2007) Ca2+ channel alpha(2)delta ligands: novel modulators of neurotransmission. Trends Pharmacol Sci 28: 75–82.
  29. 29. Li Z, Taylor CP, Weber M, Piechan J, Prior F, et al. (2011) Pregabalin is a potent and selective ligand for alpha(2)delta-1 and alpha(2)delta-2 calcium channel subunits. Eur J Pharmacol 667: 80–90.
  30. 30. Twitchell W, Brown S, Mackie K (1997) Cannabinoids inhibit N- and P/Q-type calcium channels in cultured rat hippocampal neurons. J Neurophysiol 78: 43–50.
  31. 31. Xu YR, Barbieri JT (1996) Pertussis toxin-catalyzed ADP-Ribosylation of g(i-2), and G(i-3) in CHO cells is modulated by inhibitors of intracellular trafficking. Infect Immun 64: 593–599.
  32. 32. Axelrod J, Felder CC (1998) Cannabinoid receptors and their endogenous agonist, anandamide. Neurochem Res 23: 575–581.
  33. 33. Meder W, Fink K, Zentner J, Gothert M (1999) Calcium channels involved in K+- and veratridine-induced increase of cytosolic calcium concentration in human cerebral cortical synaptosomes. J Pharmacol Exp Ther 290: 1126–1131.
  34. 34. Olivera BM, Miljanich GP, Ramachandran J, Adams ME (1994) Calcium-Channel Diversity and Neurotransmitter Release - the Omega-Conotoxins and Omega-Agatoxins. Annu Rev Biochem 63: 823–867.
  35. 35. Brum LFS, Elisabetsky E, Souza D (2001) Effects of linalool on [H-3] MK801 and [H-3] muscimol binding in mouse cortical membranes. Phytother Res 15: 422–425.
  36. 36. Leal-Cardoso JH, Silva-Alves KS, Ferreira-da-Silva FW, dos Santos-Nascimento T, Joca HC, et al. (2010) Linalool blocks excitability in peripheral nerves and voltage-dependent Na(+) current in dissociated dorsal root ganglia neurons. Eur J Pharmacol 645: 86–93.
  37. 37. Huang LP, Abuhamdah S, Howes MJR, Dixon CL, Elliot MSJ, et al. (2009) Pharmacological profile of essential oils derived from Lavandula angustifolia and Melissa officinalis with anti-agitation properties: focus on ligand-gated channels (vol 59, pg 1515, 2008). J Pharm Pharmacol 61: 267–267.
  38. 38. Re L, Barocci S, Sonnino S, Mencarelli A, Vivani C, et al. (2000) Linalool modifies the nicotinic receptor-ion channel kinetics at the mouse neuromuscular junction. Pharmacol Res 42: 177–181.
  39. 39. Batista PA, Werner MFF, Oliveira EC, Burgos L, Pereira P, et al. (2008) Evidence for the involvement of ionotropic glutamatergic receptors on the antinociceptive effect of (−)-linalool in mice. Neurosci Lett 440: 299–303.
  40. 40. Field MJ, Cox PJ, Stott E, Melrose H, Offord J, et al. (2006) Identification of the alpha(2)-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin. Proc Natl Acad Sci U S A 103: 17537–17542.