PinetoninaTM , an Intranasally Administered Essential Oil Preparation , Is Effective in Decrease of Cortisol Levels and on the Glutamate Release Modulation

Background: Most anxiety complaints are treated with pharmacological measures involving barbiturates and benzodiazepines, in which they may end up causing tolerance and pharmacological dependence. Integrative approaches such as aromatherapy are used in addition to medications to improve sleep quality and reduce anxiety. Thus, PinetoninTM, a phytocomplex obtained from a blend of essential oils aims to aid in the symptoms of stress and anxiety. Methods: The cytotoxicity of PinetoninTM was evaluated MTT assay using fibroblasts and astrocytes showed reduction in the cell viability only at high concentrations. Evaluation of intracellular calcium and determination of residual glutamate in the supernatant of astrocyte cultures showed agonist action of dihydroxyphenylglycine (DHPG) increasing linearly the concentration of intracellular calcium and the glutamate levels in the supernatants of the cultures. On the other hand, cultures of astrocytes treated with PinetoninTM showed residual glutamate levels in the supernatants reducing proportionally, as well as, intracellular calcium reduction. The determination of salivary cortisol showed a significant reduction in salivary cortisol levels in the group that received PinetoninaTM. The evaluation of the electroencephaloHow to cite this paper: Jardim, M., Amaral, F., de Souza Antunes, V.M., Rodrigues, F., de Souza, M.S.A., Siqueto, F.R., Silva, L.M., Bertolino, R.A., Cavalaro, V. and Oliveira, C.R. (2018) PinetoninaTM, an Intranasally Administered Essential Oil Preparation, Is Effective in Decrease of Cortisol Levels and on the Glutamate Release Modulation. Neuroscience & Medicine, 9, 135-149. https://doi.org/10.4236/nm.2018.93014 Received: June 12, 2018 Accepted: September 2, 2018 Published: September 5, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Background
Affecting almost 13% of the world's population, anxiety disorders are among the most common psycho-socio-behavioral problems that justify the importance and interest in the areas applied to psychopharmacology.Psychoanalytic, behavioral, genetic, biological, cognitive and psychodynamic theories are used as tools to explain the etiology of anxiety disorders since it is a disease of heterogeneous disorders [1].Most complaints related to these problems are treated with pharmacological measures involving benzodiazepines and barbiturates, in addition to other anxiolytic agents, which in the majority of cases can generate tolerance and consequently pharmacological dependence [2].Thus, to reduce the costs of pharmacotherapy and, in synergy, to complement the medicinal treatments, the essential oils appear as new therapeutic modalities.
This integrative practice has the actions as anti-inflammatory, anti-oxidants helping to reduce anxiety and stress [3].
To guarantee the emotional balance and the physical and mental estares several essential oils are obtained [4] [5].Currently, with the goals of improving sleep quality and also reducing the effects of anxiety, integrative approaches like aromatherapy are used in synergy with medications [6].
In addition, studies have demonstrated the effect of olfactory stimulation on various physiological systems, among which, anxiety-related systems.In this sense, the practice of using essential oils via the olfactory system had a positive effect on the reduction of anxiety, increasing sleep quality and stabilization of blood pressure in patients with cardiovascular disease in the intensive care unit [7].In other studies, it was possible to observe that the inhalation of essential oils reduced blood pressure in rats after stressful responses [8], and that the olfactory stimulus with essential oils, aided in the stabilization of the mood of stroke patients and who showed symptoms of anxiety [9].
However, the most explored activity of lavender essential oil is its use as an anxiolytic and as an adjuvant in the treatment of insomnia, even though there is no evidence that Lavender does not have a sedative effect, olfactory use can improve the quality of sleep reducing insomnia [12].
Foeniculum vulgare Mill, commonly known as fennel, is an aromatic plant widely cultivated in temperate and tropical regions [13].The chemical constituents of F. vulgare include essential oil, fatty acid, phenylpropanoids, monoterpenes, sesquiterpenes and coumarins.It also contains triterpenoids, tannins, flavonoids, cardiac glycosides, saponins, and other types of compounds [14].In traditional medicine, F. vulgare is used to encourage menstruation and lactation, stimulate gastrointestinal motility, relieve intestinal gas accumulation, improve eyesight, alleviate productive coughs, ease spasm, promote courage and mental strength, reduce stress and nervousness and produce calming [15].
Therefore, this work aims to evaluate a blend of essential oils in order to verify if their inhalation may contribute to the reduction of anxiety and the quality of life of the volunteers, using as indicative of these activities, the analysis of alpha (α) and beta (β) obtained by electroencephalogram.

Cell Culture
Both cell lines were kindly provided by Prof. Dr. Eduardo Finger, Coordinator of the Laboratory of Molecular Biology, Anhembi Morumbi University.The Fibroblast (CCD-1072Sk) and astrocyte (GIBCO N7805100) lines, were cultured in ISCOVE™ and DMEM/F12 medium, respectively, both with 10% fetal bovine serum, 0.292 g/L L-glutamine, 1.0 g/L D-glucose, 2.2 g/L NaHCO 3 , 10,000 IU Penicillin and 0.060 g/L Streptomycin.Cells were maintained in 25 cm 2 flasks (1 × 10 5 cells/mL) in a humid oven with 5% CO 2 atmosphere at 37˚C.In all experiments the cultures of fibroblasts and astrocytes were submitted to the cell viability test using the trypan blue dye and the reading was performed in haemocytometric chamber by light microscopy.All experiments described were performed when cell viability was equal to or greater than 95%.In all assays, the incubation time was 12 h and the concentrations of Pinetonina™ ranged from 1% -10%.Therefore, 5 × 10 4 cells/well were treated with different concentrations of Pine-tonina™ (0.5%, 1.0%, 1.5% and 2.0%).After that, 10 μL of the 5 mg/mL MTT solution (Sigma-Aldrich) was added to each well.After 4 hours, the culture was collected in 15 ml tubes and centrifuge at 1500 rpm for 5 minutes, the supernatant from each well discarded and the pellet with the crystals formed on the bottom of the plate dissolved with 100 μL of pure ethanol and then homogenized on a plate shaker for 15 minutes.Optical density was measured by the microplate reader (FlexStation® 3 multimode benchtop reader) at 540 nm.

Glutamate Supernatant
Astrocytes maintained in culture and treated according to item 2.

Statistical Analysis
The results were presented as mean + SEM (standard error of the mean).The results were submitted to statistical analysis by analysis of variance (ANOVA) of probability of type I errors was used to determine statistical significance.In all cases, P < 0.05 was taken as the level of significance.

MTT Reduction Test for Formazan
The results obtained with the MTT test are shown in Figure 1.It is possible to observe a reduction in the viability of fibroblasts and astrocytes in a dose-dependent manner.In addition, only the 10% concentration promoted a significant reduction in cellular viability when compared to the control group (untreated group), considering a value of P < 0.05.

Evaluation of Intracellular Calcium and Determination of Residual Glutamate in the Supernatant of Astrocyte Cultures
The results presented in Figure 2(a) show the agonist action of DHPG increasing linearly the concentration of intracellular calcium.The glutamate levels released in the culture medium of these astrocytes were observed, as well as the linear increase of this excitatory amino acid.In Figure 2

Determination of Salivary Cortisol
After using Pinetonin™ for 15 consecutive days, twice daily, volunteers collected saliva for determination of salivary cortisol.Figure 3 shows a significant reduction (P < 0.05) in salivary cortisol levels in the Pinetonin™ group.The groups treated with saline in the same scheme did not present a significant reduction.

Evaluation of the Electroencephalogram (Powerlab)
The evaluation of the electroencephalogram of the volunteers who received Pi-netonina™ and saline, in addition to those who did not receive any type of product, showed that Pinetonina™ influenced the alpha (α) and beta (β) waves.Volunteers treated with Pinetonina™ had a significant increase (P < 0.05) in the frequency (Hz) of the alpha waves, which are waves related to relaxation, visualization and creativity (Figure 4(a)).In Figure 4(b), it was possible to observe that the volunteers who received Pinetonina™ presented reduction in the frequency of beta waves (β), waves these related to the alert, concentration and cognition.

Discussion
Recent studies have shown that essential oils have several biological activities, including anti-inflammatory activity [17] [18] [19] [20], antimicrobial [20] [21] and sedative properties [22] [23].Essential oils such as those obtained from the Lavandula spp.what is observed in clinical practice [18].These results corroborate data found in the literature on the toxicity of some essential oils [25] [28] [29].In order to propose a possible mechanism of action of Pinetonia™, astrocyte lineage was used to assess its ability to modulate the release of glutamate, an amino acid that participates in the Central Nervous System (CNS) as an excitatory neurotransmitter [30] [31] and the mobilization capacity of intracellular calcium, a phenomenon related to the release of glutamate and several other neurotransmitters stored in vesicles [30].The results indicated that the action of the DHPG agonist on the astrocyte line increased the concentration of intracellular calcium and a proportional increase in glutamate levels was observed in the supernatant obtained from the supernatant of these cultures.On the other hand, when the astrocyte culture was previously treated with linalool, one of the main components of the essential oil of the species Lavandula spp.[25] [27] [32], which are part of the composition of Pinetonina™, we observed reduction in intracellular calcium levels, with consequent reduction of residual glutamate in the supernatant of astrocyte cultures.Thus, when analyzing the results obtained with Pinetonina™ it is possible to observe the same behavior regarding calcium mobilization in the as-

Conclusion
Based on the in vitro tests, it can be suggested that the compound Pinetonina™ is not considered cytotoxic, since only from the concentration of 10% showed to significantly reduce cell viability in cell lines evaluated.After determination of intracellular calcium and determination of glutamate in the supernatant of the astrocyte cultures, it can be deduced that Pinetonin™ was able to reduce the cal- rode solution (Inmilimolar (mM): Sodium Chloride (NaCl) 137.0;Potassium Chloride (KCl) 2.7; Clacium Chloride Dihydratade (CaCl 2 •2H 2 O) 1.4; Magnesium Chloride Hexahydrate (MgCl 2 •6H 2 O) 0.5; Sodium Dihydrogen Phosphate Monohydrate (NaH 2 PO 4 •H 2 O) 0.4; 16.0 Carbonic acid (H 2 CO 3 ), Glucose supplemented with serum bovine albumin (BSA, 0.2% w/v) and incubated with Fura 2-AM (5 micromolar (μM), 23˚C) for 1 hour under stirring to be continued.Measurements of [Ca 2+ ] i were performed on a spectrofluorimeter.Maximum and minimum rates of 340/380 were achieved by administration of the cellular permeabilizer digitonin (50 μM) and Egtazic Acid (EGTA) (4 mM) + Sodium Hydroxid (NaOH) (0.04 N) added at the end of the experiments.The functional response of the cells was assessed by the [Ca 2+ ] i alteration in response to treatment with dihydroxyphenylglycine (DHPG), Linalool, Pinetonina™ and the combination Linalol and DHPG, in the following concentrations: 1.0%, 2.5% and 5.0% compared with basal levels.

2. 5 . 2 .
Electroencephalogram (EEG)Three disposable electrodes were implanted for epidural electroencephalogram recording.The electrodes were positioned bilaterally on the frontal bone and the occipital cortex (posterior 7.4 milimeters (mm) bregma, lateral ± 5.0 mm).The reference electrode was placed posteriorly in a known anatomical region with external occipital protuberance.The cables were connected to a socket, which was secured together with the electrodes.The electroencephalogram was measured continuously before and 20 minutes after inhalation of Pinetonina™ or saline in experiment 1 and in the case of the control experiment the recording was performed without inhalation of any agent.The electroencephalographic signal was digitized online at a sampling rate of 200 Hertz (Hz) and subjected to off-line spectral analysis.The power spectra were calculated for consecutive 2.56 s intervals and 0.39 Hz frequency compartments using the labchart analysis system.In this study, the global electroencephalographic (0 -30 Hz) frequency band was divided into two frequency bands: α (8 -13 Hz) and β(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).
one way, followed by Tukey test.Values of P < 0.05 were considered significantly different.The analysises were performed in the program GraphPad Prism version 5.0.The statistical analyzes used in the trials involving the powerlab were expressed as percentage control of the baseline values and represented by means Neuroscience & Medicine ± SEM.The significance of the differences between the mean values of the EEG experiment was determined by repeated measures of variance analysis (ANOVA) followed by the Student-Newman-Kewls (SNK) test for multiple comparisons.All statistical analyzes were performed using the Statistical Package for the Socail Science (SPSS) 22.0 program (SPSS, Chicago, IL), and a 5% (b), we observed that linalool, one of the main components of the Pinetonina™ blend, promoted reduction in intracellular calcium levels.When looking at the astrocytes treated with Pinetonina™ (Figure 2(c)), it is possible to note that intracellular calcium levels are reduced starting from the 2.5% concentration.Finally, the 2D figure indicates that previous treatment with linalol reversed the increase of intracellular calcium and residual glutamate in the culture medium of the astrocytes.

Figure 1 .
Figure 1.Results of cell proliferation through the MTT reduction assay after 24 hours exposure with different concentrations of Pinetonin™.Prior to the start of the tests, the cells were depleted of fetal bovine serum.(*) P <0.05-significant in relation to the control, ANOVA, Tukey.Trials performed in triplicate.GraphPad Prism V.5.0.

Figure 2 .
Figure 2. Levels of intracellular calcium and glutamate residue in the supernatants of astrocyte cultures.(a) cultures treated with the glutamatergic receptor agonist, DHPG; (b) cultures treated with linalool; (c) cultures treated with Pinetonin™; (d) cultures previously treated with linalool and then with DHPG.(*) P < 0.05-significant in relation to the control, ANOVA, Tukey.Trials performed in triplicate.GraphPad Prism V.5.0.

Figure 3 .
Figure3.Variation of the level of salivary cortisol between groups of volunteers who received Pinetonina™, saline and without treatment.The group treated with Pinetonina™ obtained lower mean (%) when compared to the control group (without treatment) and when compared to the group treated with saline.(*) P < 0.05-significant in relation to the control, ANOVA, Tukey.Trials performed in triplicate.GraphPad Prism V.5.0.

Figure 4 .
Figure 4. Frequency pattern of alpha and beta brain waves.(a) significant increase in the alpha wave frequency of the Pinetonin™ group when compared to the saline and control groups (without intranasal application); (b) reduction in beta-wave frequency of the Pinetonin™ group when compared to saline and control groups.(*) P < 0.05-significant in relation to the control, ANOVA, Tukey.Trials performed in triplicate.GraphPad Prism V.5.0.
obtained with linalool, besides reducing the residual glutamate in the supernatant of the cultures.Studies showing the intracellular calcium modulation capacity with consequent release of glutamate by astrocytes allow us to infer the hypothesis that components present in the composition Pinetonina™ could reduce, albeit partially, the release of glutamate allowing a greater action of the inhibitory neurotransmitter Gamma-Amino Butyric Acid (GABA) responsible for part of the sedative action in the CNS[33] [34].In order to evaluate in vivo the effects of stress-related and relaxation-related Pinetonin™, we performed salivary cortisol dosing of the volunteers after 15 consecutive days of use of the product.When compared to control volunteers (who received no treatment) and placebo volunteers (saline), we observed a significant reduction in salivary cortisol levels.This study shows the probably indirect influence of calcium and glutamate on the release of cortisol through the modulation of release of Corticotropin Releasing Hormone (CRH)[26] [33][35] [36], which would ultimately signal the release of adrenocorticotrophic hormone (ACTH) for the production of cortisol in the adrenal cortex.The idea of a lower production of cortisol would be related to the greater ease of melatonin release, since the latter hormone is elevated when cortisol is reduced[37] [38], thus aiding in sleep quality[39] [40][41].Finally, the electroencephalogram evaluation of the volunteers showed that Pinetonina™ influenced the alpha (α) and beta (β) waves.The results showed that volunteers treated with Pinetonina™ had a significant increase (P < 0.05) in the frequency (Hz) of alpha waves, which are waves related to relaxation, visualization and creativity[41] [42], whereas volunteers who received Pinetonina™ presented a reduction in the frequency of beta waves (β), these waves related to the alert[42] [43][44].
cium concentration and the release of glutamate (excitatory neurotransmitter) into the supernatant of the astrocyte cultures, suggesting amodulating activity between excitatory and inhibitory systems in the CNS.Regarding the in vivo tests, the salivary cortisol dosage of the volunteers that made intranasal application of Pinetonina™, allows us to conclude that the levels of this hormone, which in situations of stress is high, and has their values reduced.As melatonin levels begin to rise as cortisol levels begin to decline, we may suggest that Pinetonin™, in daily stress situations where cortisol levels begin to decay much later than normal, would reduce cortisol circulating, facilitating the increase of melatonin, making the patients who use Pinetonina™ have better quality of sleep and less stress.Associated with the reduction of salivary cortisol, patients who underwent M. Jardim et al.DOI: 10.4236/nm.2018.93014145 Neuroscience & Medicine intranasal application showed an increase in the CNS alpha wave pattern on the electroencephalogram, indicating that Pinetonin™ may promote relaxation in individuals using the compound, while reducing beta brain waves, responsible for the alert, and other aspects of the CNS.Thus, it was possible to prove that the inhalation of Pinetonina™ reduces the anxiety, providing an increase in the quality of life of the volunteers.Funding The present study was supported by the Institute of Osmology and Essential Oils of Brazil, the Anhembi Morumbi University of Brazil and the Group of Fitocomplexes and Cellular Signaling from School of Health Science, Anhembi Morumbi University of Brazil.
concentrations of glutamate by the ordinary least squares equation y = 0.073X + 0.122 was used to quantify the results.
and analyzed by the glutamate assay kit (Biovision) according to the manufacturer's instructions.A standard curve generated by different concentrations of glutamate in DMEM-Dulbecco's Modified Eagle Medium-was used to calcu-late the plication, being this group the one used to take basal electroencephalogram waves.After the application of the intranasal doses (puffs), either Pinetonina™ or saline, each of the volunteers waited in a segregated place, without intense noise and without excessive light for 30 minutes, and then underwent electroencephalogram evaluation.The group that received no intranasal application performed the same procedure as the groups receiving either Pinetonina™ or saline.