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Exploring Pharmacological Mechanisms of Lavender (Lavandula angustifolia) Essential Oil on Central Nervous System Targets

Abstract

Lavender essential oil is traditionally used and approved by the European Medicines Agency (EMA) as herbal medicine to relieve stress and anxiety. Some animal and clinical studies reveal positive results in models of anxiety and depression although very little research has been done on molecular mechanisms. Our work consisted of evaluating the effects of lavender (Lavandula angustifolia) essential oil on central nervous system well-established targets, such as MAO-A, SERT, GABAAand NMDA receptors as well as in vitro models of neurotoxicity. The results showed that lavender essential oil and its main components exert affinity for the glutamate NMDA-receptor in a dose-dependent manner with an IC50 value of 0.04 μl/mL for lavender oil. In addition, lavender and linalool were also able to bind the serotonin transporter (SERT) whereas they did not show affinity for GABAA-benzodiazepine receptor. In three different models of neurotoxicity, lavender did not enhance the neurotoxic insult and improved viability of SH-SY5Y cells treated with hydrogen peroxide. According to our data, the anxiolytic and antidepressant-like effects attributed to lavender may be due to an antagonism on the NMDA-receptor and inhibition of SERT. This study suggests that lavender essential oil may exert pharmacological properties via modulating the NMDA receptor, the SERT as well as neurotoxicity induced by hydrogen peroxide. Keywords: essential oils, central nervous system, lavender, Lavandula angustifoliaLavandula officinalis, SH-SY5Y cells, glutamate receptor Go to:

Introduction

Essential oils have a long tradition in pharmaceutical sciences as natural products with pharmacological, cosmetic, agrochemical and nutritional applications (Bakkali et al., 2008). The use of EO in form of aromatherapy or phytotherapy is widely extended, some of them being used as agents to relieve anxiety and stress (Setzer, 2009). Phytotherapy consists of the use of medicinal plants in order to prevent, cure or threat illnesses. Aromatherapy can be understood as a subdivision of phytotherapy and defined as the use of essential oils regarding therapeutic effects. These products have been used for centuries and are accepted in traditional or modern healthcare systems of medicine. Medicinal plants are widely used for the treatment of central nervous system disorders (Wheatley, 2005) but in some cases there is still lack of preclinical and clinical studies.

Central nervous system disorders have a great impact in society due to a general aging process of the population as well as lifestyle. Stress is one of the most prevalent psychological disorders in developed countries leading to other clinical features, such as anxiety, insomnia or depression.

Benzodiazepines (BZD) and selective serotonin reuptake inhibitors (SSRIs) are highly prescribed as anxiolytic and antidepressant drugs, respectively. BZD, such as diazepam, lorazepam or alprazolam produce calming effects via binding to GABAA receptors, but they may also produce somnolence and cognitive impairment as adverse drug reactions. SSRIs (e.g., fluoxetin, paroxetin, citalopram) are prescribed as antidepressants because they are able to selectively block the serotonin transporter (SERT), but side effects include sexual dysfunction and neuropsychiatric disorders, such as suicide tendencies and sleep disorders. Both groups of medicines are also involved in withdrawal and “rebound effects” as a result of discontinuing their administration.

Certain EO are being used as anxiolytic remedies and the administration mode can be orally but also by inhalation or combined with massage. One of the most popular essential oils for mental disorders and anxiety is lavender (Lavandula angustifolia Miller or Lavandula officinalis Chaix). Lavender essential oil can be considered as one of the best-seller over the counter herbal remedies for anxiety, stress and depression. Studies reveal high content of linalool and linalyl acetate (Da Porto et al., 2009) and international organizations, such as the World Health Organization (WHO), the European Scientific Cooperative on Phytotherapy (ESCOP) or the European Medicines Agency (EMA) approve this medicinal plant to relieve stress, restlessness and anxiety (Community)1.

The growing awareness of the adverse effects of central nervous system drugs has led to develop new strategies and safer pharmacological agents in mental health. Enzymes, such as monoamine oxidase (MAO), proteins, such as the SERT and ligand-gated ion channels, such as GABAA and NMDA receptors are therapeutic targets in neuropharmacology. With the aim to contribute to evidence-based herbal medicine we have studied the effects of lavender essential oil on pharmacological targets involved in anxiolytic and antidepressive properties as well as in vitro models of neurotoxicity.Go to:

Methods

Lavender essential oil (LEO) and chemicals

Pure lavender (L. angustifolia) essential oil was kindly supplied by Pranarom International (Belgium). Isolated monoterpenes (linalool, linalyl acetate) were purchased from Fluka. Enzymes, proteins and reagents were acquired from Sigma. Linalool and linalyl acetate were also tested when lavender essential oil had a clear and significant activity in the assays.

Chemical profile by GC-MS

Although the essential oils are chemically characterized by Pranarom International, LEO was analyzed in the laboratory by GC-MS on an Agilent 6890N Network GC system coupled to a 5973 Network Mass Selective Detector, accelerating voltage -69.9 eV, recoding masses of 35.00–400.00. GC conditions: injector temperature: 150°C; temperature programme: start 50°C, 20°C/min to 300°C; column: HP5MS (5% phenylmethylsiloxane) capillary, 30.0 m × 250 μl × 0.25 μm nominal. Carrier gas: helium at 1.0 ml/min. A NIST library was used for comparison of MS data.

Animals and brain membrane homogenates

Adult male Sprague Dawley rats were obtained from Taconic (Denmark). Ethical permission for the studies was granted by the Animal Welfare Committee, appointed by the Danish Ministry of Justice, and all animal procedures were carried out in compliance with the EC Directive 86/609/EEC and the Danish laws regulating experiments on animals. Rats were put down by competent staff, the heads were separated from the body and the brains were removed whereas cerebellum was discarded. The cortex were weighed and homogenized with an Ultra-Turrax using different buffers at 4°C and the tissue preparation in each case was carried out as earlier described (Ransom and Stec, 1988). The tissue homogenates were resuspended and stored in aliquots at −80°C until use.

Bioassays regarding serotonergic targets

Monoamine oxidase-a inhibition (MAO-A Assay) 

The bioassay was performed in a 96-well microplate (Saaby et al., 2009). Each well contained 50 μl of essential oil dilution or DMSO as blank (making a final concentration in the wells of 0.002, 0.01, 0.02, and 0.1%), 50 μl of chromogenic solution (0.8 mM vanillic acid, 417 mM 4-aminoantipyrine and 4 U/ml horseradish peroxidase in potassium phosphate buffer pH 7.6), 100 μl of 3 mM tyramine and 50 μl of 8 U/ml MAO-A. Absorbance was read at 490 nm every 5 min for 30 min. Background interferences were deducted in the same way described above but without MAO enzyme. Data was analyzed using GraphPad Prism. IC50 values could not be obtained because the higest tested concentration did not reach 50% of MAO inhibition. Clorgyline was used as positive control.

Serotonin transporter assay ([3H]-citalopram binding assay) 

The assay was performed on the basis of the method from Nielsen et al. (2004). 25 μl of three different essential oil concentrations were mixed with 50 μl of 4 nM [3H]-Citalopram and 225 μl of rat cortex suspension making a final concentration of the essential oils in the assay of 0.08, 0.4, and 0.8%. All components were previously dissolved in buffer (50 mM Tris-base; 120 mM NaCl: 5 mM KCl at pH 7.5). Plastic tubes were placed on an ice bath, the reagents were added and the tubes were mixed and then left at room temperature (approximately 22°C) for 2 h. After incubation, 5 ml of ice cold buffer were added to the samples and they were filtered through GC-50 Advantec glass filters under vacuum and immediately washed once with additional 5 ml of ice cold buffer. Control test tubes (with buffer instead of EO) and blind tubes (with 25 μl of 120 μM paroxetine instead of EO) were done in every run in order to determine total and unspecific binding. The amount of radioactivity was determined transferring the glass filters into scintillation tubes and adding 4 ml of Ultimo Gold XR. The scintillation tubes were placed in the dark for 30 min before they were measured on a Tri-CARB 2100 TR analyzer. Three independent experiments were performed in triplicates. Non-radioactive citalopram was used as reference. Linalool and linalyl acetate were tested. The specific binding of [3H]-citalopram was determined using the formula: % binding = [dpm(essential oil)-dpm(unspecific binding)/dpm(total binding)-dpm(unspeficic binding)] × 100.

Bioassays on ionotropic receptors

Affinity for GABAA receptor ([3H]-Ro 15-1788 binding assay) 

The membrane preparation was washed with ice-cold buffer (50 mM Tris-citrate pH 7.1). The suspension was centrifuged at 0–4°C for 10 min at 27,000 × g. The pellet was resuspended in Tris-citrate buffer (2 mg original tissue per ml) and used for the assay. 25 μl of 3H-Ro 15-1788 (flumazenil) was added to 25 μl of test solutions (10, 1, and 0.1 mg/ml) and 500 μl of membrane preparation. Total and unspecific binding was measured using buffer or diazepam (1 μM assay final concentration). After incubation for 40 min in an ice bath, 5 ml of ice-cold buffer was added to the samples and poured onto Adventic glass fiber filters (GC-50) under vacuum, and immediately washed with another 5 ml of ice-cold buffer. The amount of radioactivity of the filters was measured by conventional liquid scintillation counting using Ultimo Gold XR as scintillation fluid. Clonazepam was used as positive control.

Affinity for NMDA receptor ([3H]-CGP39653 binding assay) 

Affinity for native NMDA receptors was determined using 2 nM [3H]-CGP 39653 (Sills et al., 1991) with some modification. On the day of the assay, frozen membranes were quickly thawed and homogenized in 30 volumes of ice-cold Tris-HCl buffer, pH 7.4 (50 mM containing 2.5 mM CaCl2), and centrifuged (48,000 × g for 10 min). This step was repeated three times. The final pellet was re-suspended in ice-cold buffer, corresponding to approximately 0.4–0.5 mg protein/ml. Binding was carried out in aliquots consisting of 25 μL [3H]-CGP 39653, 25 μL test solution, and 200 μL membrane suspension and incubated at 0° for 60 min. Non-specific binding was determined using 1 mM (S)-Glu. Binding was terminated by filtration through Whatman GF/C filters using a 96-well Packard Filter-Mate Cell Harvester and filters were washed with 3 × 250 μL of ice-cold buffer. After drying, 30 μL Microscint 0 (Perkin-Elmer) per well was added and the filter was counted on a Topcounter (Perkin-Elmer). Linalool and linalyl acetate were also tested in this assay.

Neuroprotection on SH-SY5Y cells

SH-SY5Y neuroblastoma cell culture 

Cells were cultured in DMEM (Gibco, ref. 41966-029) containing phenol red (PR), L-Glutamine (1 mM) and sodium pyruvate (1 mM) and was supplemented with 10% FBS (Gibco), penicillin (100 U/mL, Gibco) and streptomycin (100 U/mL, Gibco). Cells were maintained at 37°C in saturated humidity (5% CO2). Non-differentiated cells were plated on 48-well plates (Corning), at a density of 5 × 104 cells per well and were used 48 h after seeding.

Aβ25–35solublespecies preparation 

25–35fragment was purchased from Sigma-Aldrich laboratories (ref. A4559-1MG). 1 mg of Aβ25–35 fragment was dissolved in 1 mL of type II water and was frozen at -20°C until further use. Different concentrations of Aβ25–35 (5, 10 and 15 μM) were incubated in DMEM phenol red free (Gibco, 31053-028) at 37°C for 3 days to obtain the oligomeric forms.

Cell treatment and MTT cell viability assay 

SH-SY5Y cells were treated with either different LEO concentration (0.05, 0.1, 0.5, and1 μL/mL) or incubation time (0, 2, and 24 h), followed by hydrogen peroxide (100, 200, 400, 800, and 1,600 μM), malonate (0 or 50 mM) or Aβ25–35(5, 10, and 15 μM).

Cell viability was examined by the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay is a colorimetric assay for measuring the activity of cellular enzymes in living cells in response to potential toxic. After the cell treatment, culture medium was replaced by a solution of 5 mg/mL MTT (Sigma-Aldrich) in DMEM (Gibco, 31053-028). Cells were incubated with MTT solution for 2 h in the cell incubator (5% CO2 and 37°C). Then, the MTT solution was discarded and DMSO was added to the wells. Aliquots were transferred to a 96-well plate, and absorbance was measured at 595 nm in a plate reader. Results were expressed as percentages of non-treated control cells.

Statistical analyses

Data are expressed as mean ± SE (figures) or as mean ±SD (tables) of at least three independent experiments performed in different days and in triplicates. GraphPad Prism was used to calculate IC50 values and to detect significant differences. Student t-test or ANOVA was performed for data analysis.Go to:

Results

Chemical profile by GC-MS

According to the GC-MS analyses, lavender essential oil mainly contained the following monoterpenes: linalyl acetate (52.1%), linalool (37.4%), geranyl acetate (5.4%), and β-caryophyllene (5.1%).

Monoamine oxidase-a inhibition (MAO-A assay)

Inhibition of MAO A was not detected (data not shown).

Serotonin transporter assay ([3H]-citalopram binding assay)

The effects of LEO on the SERT are displayed in Table ​Table1,1, showing moderate activity in this assay (citalopram IC50 value was 1.3 nM). LEO significantly displaced 3H-citalopram from binding to the SERT in a dose-dependent manner, which means that LEO might have an antidepressant-like effect via this specific transporter. This effect was also detected for linalool, one of the main constituents of lavender essential oil (Figure ​(Figure1),1), but not for linalyl acetate.

Víctor López,1,2,*Birgitte Nielsen,2Maite Solas,3Maria J. Ramírez,3 and Anna K. Jäger2Author information Article notes Copyright and License information Disclaimer This article has been cited by other articles in PMC. Go to:

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