Alpha-terpineol: evaluation and pharmacological screening as an antidepressant agent

Objective: To evaluate the possible antidepressant effects of alpha-terpineol in rodents. Material and Methods: Depression levels were analyzed by comparing the total immobility time presented by the animals of the experimental groups in the test session, using the Forced Swimming Test and the Tail Suspension Test. The parameters of locomotion (central, peripheral and total) and motor coordination were evaluated in the Open Field Test and in the Rota Rod Test, respectively. In the second stage, the involvement of the noradrenergic system in the antidepressant action of alpha-terpineol in Forced Swimming Test was investigated. Results and Discussion: After performing the experimental tests, it was observed that the animals that received alpha-terpineol had reduced immobility time in Forced Swimming Test and Tail Suspension Test, compared to the other groups. In the Open Field Test and Rota-rod, the mice showed, respectively, good exploratory activity and motor coordination during the tests. In addition, the Research, Society and Development, v. 10, n. 11, e191101119571, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19571 2 study of the Noradrenergic System proved to be a promising mechanism used during its antidepressant action. Conclusion: In view of the results of the experimental tests, alpha-terpineol presented similar responses to those found in other monoterpenes investigated in the literature. Thus, it is shown as a promising antidepressant to be used clinically in humans, with less side effects and low production cost.


Introduction
Depression is a common disorder worldwide, and it is estimated that more than 300 million people suffer from it. The condition is different from the usual fluctuations in mood and short-term emotional responses to the challenges of everyday life Pan American Health Organization -PAHO (2018). In addition, it is associated with anxiety, decreases the patient's quality of life, and may present degrees of suffering ranging from sadness to thoughts of death (Pimenta, Seixas, Gregório, & Santos, 2016).
Although intense research on depression is being carried out, the etiology and pathogenesis of this disease are still unclear. There are preclinical and clinical studies suggesting that monoaminergic neurotransmitters such as serotonin (5-HT), norepinephrine (NA) and dopamine (DA) play an important role in the pathophysiology of the central nervous system (CNS) (Możdżeń, Wąsik, Romańska, Michaluk, & Antkiewicz-Michaluk, 2017).
According to Tundo, de Filippis and Proietti (2015), about 30% of depressed patients evaluated in their research do not respond adequately to initial drug therapy. As found in the study by Ibanez (2014), which reports the difficulty of these Research, Society andDevelopment, v. 10, n. 11, e191101119571, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19571 3 individuals adhering to drug treatment, and a considerable portion of the population has a low level of knowledge in relation to the dose and frequency of administration.
In this perspective, the use of plant species for therapeutic purposes, for the treatment, cure and prevention of diseases is one of the oldest means practiced by medicine. The production of drugs of plant origin involves ethnopharmacological surveys, phytochemical and pharmacological studies. In addition, biological investigations are essential and animal models of anxiety and depression have played relevant roles in the research process for new drugs (Lima et al., 2019).
Alpha-terpineol, a volatile monoterpenoid alcohol, is the main component of essential oils from various species of aromatic plants, such as Origanium vulgare L. and Ocimum canum Sims, widely used for medicinal purposes. Studies indicate that it can also be isolated from a variety of sources, such as cajuput, pine and petitgrai oils, as well as having a wide range of biological properties, including cardiovascular and antihypertensive effects; antioxidant, antinociceptive, antiulcerative, anticonvulsant and sedative activity (Khaleel, Tabanca, & Buchbauer, 2018).
Faced with so many therapeutic possibilities presented by this monoterpene, this work has the general objective of elucidating the possible antidepressant action exerted by alpha-terpineol.

Nature of the Study
This study corresponds to an experimental research with a quantitative and qualitative approach. It is classified as explanatory in terms of objectives, as it proposes to investigate the cause and effect relationships of data collected in the laboratory (Pereira, Shitsuka, Parreira & Shitsuka, 2018).
According to Yin (2015), quantitative and qualitative studies can be complementary in order to clarify the phenomenon studied.

Study Scenario
The research was carried out at the Biotechnology and Biodiversity Research Center (NPBio, as per its Portuguese acronym) at the State University of Piaui (UESPI, as per its Portuguese acronym) and at the Pharmaceutical Technology Center (NTF, as per its Portuguese acronym) at the Federal University of Piaui (UFPI, as per its Portuguese acronym).

Animals
Albino mice (Mus musculus), Swiss variety, adult males, weighing between 25 and 30 g, were placed in acrylic cages and kept at 22 ± 2 ° C with water and food ad libitum, kept in a light / dark cycle 12:12 hours (7: 00-19: 00 h). The animals came from the NPBio Vivarium and, after the experimental procedures, euthanized with sodium Pentobarbital overdose, 150mg / kg, intraperitoneally (i.p.) associated with 10 mg/kg of lidocaine.

Forced Swim Test (FST)
This test was based on the model by Porsolt, Le Pichon and Jalfre (1977). The animals were immobilized for a certain period (duration of six minutes) keeping a record of their behavior. A tank of 22 cm in diameter and 40 cm in height containing fresh water at 26 ± 1ºC up to half was used, so that when immersed, the mouse's tail did not touch the bottom of the container.
The animals, divided into 4 groups of 6 mice (n = 6 / group) and treated as previously described, were placed, one at a time, in the tank. The immobility time was recorded in seconds for 5 minutes and the animal was considered immobile when it remained floating in the water, making only the smooth movements necessary to keep its head on the surface ( Figure 2).

Tail Suspension Test (TST)
The test protocol was performed based on the work of Steru, Chermat, Thierry and Simon (1985). In this experiment, the mice were isolated on a platform, being suspended by the tail at a height of 50 cm from the ground, using an adhesive tape placed approximately 1 cm from the tip of the tail (Figure 3). Thus, the total immobility time of the animal in the last 4 minutes of a total of 6 minutes of testing was analyzed. It is proposed that substances with antidepressant activity decrease the animals' immobility time in this test, without altering their locomotor activity. Source: Personal archive (2021).

Open Field Testing (OFT)
This test was initially proposed by Archer (1973). It was used a transparent glass box with a black background and dimensions 30x30x15cm divided into nine equal quadrants ( Figure 4). Each animal was placed in the center of the box before the test, allowing them to explore it for five minutes, with only the number of squares crossed by the animal (used as a parameter of spontaneous locomotor activity and the number of surveys performed) being recorded. Between each experiment, the box was sanitized with 5% ethanol to remove feces and urine from the animals.

Rota-rod test
The adapted test by Gonçalves et al. (2008) by Montenegro, Sena, Barbosa Filho and Almeida (2010) and Nogueira Neto et al. (2012) measures the effect of muscle relaxation or impaired motor coordination produced by compounds in animals.
The mice were placed (with four legs) on a bar 2.5 cm in diameter, raised 25 cm from the floor, in a rotation of 16 rpm, for a period of three minutes ( Figure 5). Thus, the time spent on the rotating bar in seconds was considered and the number of falls was counted, with a maximum of three renewals, following the protocol of previous studies.

Involvement of the noradrenergic system
All compounds were dissolved in saline (NaCl 0.9%) and administered i.p., except alpha-terpineol, fluoxetine and distilled water, which were administered orally (gavage). In order to investigate the participation of the noradrenergic system in the antidepressant effect of alpha-terpineol, the animals in Group 1 were pre-treated with 1mg/kg yohimbine, ip, α2adrenergic receptor antagonist) and after thirty minutes received an effective dose of alpha-terpineol. Group 2 animals were treated with propranolol (2 mg/kg, i.p., β-adrenergic receptor antagonist) and after thirty minutes received an effective dose of alpha-terpineol. Both groups (1 and 2), after 1 hour, were submitted to FST.
During the last stage, the mice in group 6 received only the effective dose of alpha-terpineol (by gavage); those in the positive control group received an effective dose of fluoxetine (20mg/kg, v.o.) and those in the negative control group were treated with an appropriate vehicle of distilled water (0.1 mL/10g of weight, v.o.). After 60 minutes, the animals were submitted to FST. Research, Society and Development, v. 10, n. 11, e191101119571, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19571 7

Statistical analysis of the data
The data were analyzed using the GraphPad Prism 7.0 program using One-Way ANOVA followed by the Tukey test.
For all tests, a significance level of 5% was considered. The results were expressed as means and standard deviation and considered positive when there was a statistically significant increase (p <0.05).

Ethical and legal aspects
This work was developed following the ethical principles of animal experimentation, in accordance with criteria

Forced Swimming
The FST results shown in Figure 6 and Table 1 demonstrate that there was a significant difference between the groups Fluoxetine (p <0.01), TEP 50 mg/kg (p <0.01) and TEP 100 mg/kg (p <0.001) in relation to the negative control. That is, the animals reduced the immobility time after receiving the doses of this monoterpene, showing a probable antidepressant action. The results were expressed as mean ± standard deviation of the immobility times of the animals in the forced swimming test by groups. ***p<0.001 and **p<0.01 when compared to the negative control group. Statistical analysis was performed using the ANOVA variance test, followed by the Tukey test. Source: Personal archive (2021).

Subtitle:
NC: Negative Control FLX: Fluoxetine 20 mg/kg TEP 50: Alpha-Terpineol 50 mg/kg TEP100: Alpha-Terpineol 100 mg/kg Thus, preclinical experimental studies using mice and behavioral testing protocols are of paramount importance to assess the effectiveness of substances with potential antidepressants. In one of these trials, there was a reduction in the immobility time of mice in FST, when they received Proposis cineria at a dose of 200 mg/kg compared to the control group, demonstrating significant antidepressant activity and relaxation of skeletal muscle (George, Joseph, & Sharma, 2012). The

Tail suspension
In the TST, it can be observed that the groups that received terpineol in the doses 50 mg/kg (p <0.01) and 100 mg/kg (p <0.1) showed statistical difference in relation to the control group (Figure 7). During the suspension experiment, the mice showed greater mobility, with the probable antidepressant characteristic being noticeable without altering their locomotor activity (Table 2). The results were expressed as mean ± standard deviation of the immobility times of the animals in the Tail Suspension test by groups. ***p<0.001 and *p<0.05 when compared to the negative control group. Statistical analysis was performed using the ANOVA variance test, followed by the Tukey test. Source: Personal archive (2021). In this perspective, the work of Dougnon and Ito (2020) proposed to investigate the substances 1,8-and 1,4-cineol, widely used for tracking antidepressant drugs in animal models, through the tests of forced swimming and suspension by tail.
In these tests, the rats are forced to swim or are hung by the tail in an unavoidable situation; first they demonstrate vigorous activity trying to escape from the threatening environment and, finally, they remain immobile as a symptom of behavioral despair. Substances that decrease immobility time are known to demonstrate antidepressant properties in humans.

Open Field
Exploratory/locomotor activity and motor coordination were evaluated by the open field and Rota-rod tests, respectively. These tests are classic animal models to track the activity of the central nervous system and provide information on anxiety, depression, sedatives, seizures and myorelaxing activities (Diniz et al., 2019).
According to the information in Figure 8, the groups that received alpha-terpineol doses (50 mg/kg and 100 mg/kg) covered a greater number of quadrants in the time of 5 minutes (mean ± standard deviation 61.40 ± 22, 26 and 60.60 ± 23.37, respectively), in relation to the negative and positive control groups (Table 3). It is noticed that this monoterpene acted to improve the spontaneous locomotor activity of animals, similar to that observed in the action of conventional antidepressants.
In addition, the means ± standard deviation of the number of withdrawals was also more significant in the groups treated with alpha-terpineol (Table 4).
In contrast, in the results by Abouhosseini Tabari, Hajizadeh Moghaddam, Maggi and Benelli (2018) the administration of P. roseum essential oil, diazepam and buspirone did not significantly change the general locomotor activity (p> 0.05), but the number of breeding and cleaning behavior was significantly reduced in diazepam, buspirone, P. roseum 20 mg, and mice treated with P. roseum 50 mg. That is, the exploratory activity of the rodents was not satisfactory, as identified with alpha-terpineol. The results were expressed as mean ± standard deviation of the number of invasions of animals in the Open Field test, by groups. In this protocol there was no statistical difference between the groups tested. Statistical analysis was performed using the ANOVA variance test, followed by the Tukey test Source: Personal archive (2021).

Rota-rod
The findings found in the rota-rod test, shown in Figure 9 and Table 5, revealed that there was no statistical difference between the negative control, positive control (fluoxetine), TEP 50 mg/kg and TEP 100 mg/kg groups. Thus, the tested substance did not cause loss of performance and motor coordination of the animals. This response is of paramount importance in relation to the effectiveness of alpha-terpineol and the adverse effects that it could cause. Results were expressed as mean ± standard deviation of the time (s) on the rotating bar during the Rota-Rod test, by groups. In this protocol there was no statistical difference between the groups tested. Statistical analysis was performed using the ANOVA variance test, followed by the Tukey test Source: Personal archive (2021).  In addition, the absence of adverse drug combination effects is also suggested by the results of the rota-rod test, which is used as a standard test for estimating motor coordination. This worked showed that ketamine at a dose of 3 mg/kg, injected separately or in combination with LY341495 (0.3 mg/kg), did not significantly alter the motor coordination of rats (Podkowa, Pochwat, Brański, Pilc, & Pałucha-Poniewiera, 2016).
However, another study showed that the administration of Nymphaea Lotus extract at 180 mg/kg in mice increased the freezing time and reduced the total crossing, indicating that the substance may have sedative or muscle relaxant activities.
When combined with the lack of motor coordination observed in the rota-rod test, this seems consistent (Fajemiroye, Adam, Zjawiony Jordan, Alves, & Aderoju, 2018).

Propranolol 2 mg/kg
The results indicate that Propranolol, a beta-adrenergic blocker, when associated with alpha-terpineol 50 mg/kg increases the immobility time of animals in FST ( Figure 10). It is argued that the drug acts by reducing the antidepressant activity in terpineol by blocking beta-adrenergic receptors, preventing the monoterpene from interacting with them, increasing the values of mean ± standard deviation, when compared to the other groups (Table 6). The results were expressed as mean ± standard deviation of immobility time in the Forced Swimming test, by groups. In this protocol there was no statistical difference between the groups tested. Statistical analysis was performed using the ANOVA variance test, followed by the Tukey test. Source: Personal archive (2021).  Research, Society andDevelopment, v. 10, n. 11, e191101119571, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19571 13 Propranolol or propranolol hydrochloride, chemically recognized as 1-isopropylamino-3-(nafityloxy) -2-propanol, is a non-selective β-adrenergic antagonist drug that interacts with β1 and β2 receptors with the same affinity (Godoy, Kummrow, & Pamplin, 2015).
Propranolol is a non-selective beta-adrenoreceptor antagonist, also classified as a class II antiarrhythmic agent. It exerts its response by competitively blocking beta-1 and beta-2 adrenergic stimulation in the heart, which is typically induced by epinephrine and norepinephrine (Rehsia & Dhalla, 2010).

Yohimbine 1 mg/kg
According to the results, there was no statistical difference between the groups that received Fluoxetine, Yohimbine 1mg/kg and Yohimbine combined with Terpineol 50 mg/kg compared to the control group ( Figure 11). However, it was observed that this association increased the immobility time of mice treated with alpha-terpineol, suggesting that terpineol acts on alpha 2 adrenergic (α2-adrenergic) receptors. Thus, when yohimbine blocks these receptors, terpineol does not bind to them, ceasing to act as a possible antidepressant. The results were expressed as mean ± standard deviation of immobility time in the Forced Swimming test, by groups. In this protocol there was no statistical difference between the groups tested. Statistical analysis was performed using the ANOVA variance test, followed by the Tukey test. Source: Personal archive (2021).
In this sense, the values of the mean ± standard deviation of the combination Yohimbine + Terpineol 50 mg/kg were similar to the negative control (Table 7). This demonstrates that the animals spent more time immobile and with depressive behavior during FST. Thus, a similar response was observed in pretreatment with yohimbine (α2 adrenoceptor antagonist), which blocked the effect of the extract of the species Mangifera indica (Anacardiaceae) of the antidepressant type, suggesting the involvement of the pre-synaptic α2 autoreceptor activity in its effect (Ishola, Awodele, & Eluogu, 2016). This same effect was shown in the studies by Savegnago et al. (2007) and Posser et al. (2009), in which the antidepressant activity of some organocalcogens, such as diphenyl diselenide and ebselen, was blocked with previous an///tagonist treatment, such as prazosin and yohimbine.
Outside FST, researchers realized that the decrease in exploration in the Labyrinth Test and the increase in immobility in OFT of mice treated with yohimbine is characterized as reminiscent of the freezing/anxiety behavior. When associated with fear, it led them to remain without further exploration. In addition, yohimbine produced a clear anxiogenic effect in the open field, plate with orifice and chiaroscuro tests (Pitzer, La Porta, Treede, & Tappe-Theodor, 2019).

Conclusion
Thus, alpha-terpineol showed favorable responses as an antidepressant agent, this effect being similar to that of the reference antidepressants already used in clinical practice. Based on the investigation of the noradrenergic pathway, the responses presented by the testing of mechanisms with propranolol and yohimbine were promising. Thus, to exert its antidepressant effect, alpha-terpineol can act by increasing the levels of norepinephrine in the body.
In this sense, it is possible to perceive the benefits caused by this monoterpene during the screening of experimental protocols. In view of this and other actions already described in the literature, such as anti-inflammatory, anticonvulsant and antioxidant, alpha-terpineol proved to be an excellent pharmacological candidate.
Finally, we suggest the investigation of tests that verify the action of alpha-terpineol in the body, as well as other mechanisms of action related to its activity, in order to alleviate the limitations brought about by the work. Furthermore, the next steps in this study are: to evaluate the possible side effects caused by alpha terpineol, its best dosage and form of presentation; in order to provide security for a probable clinical use in humans.