Anxiolytic-like Effect of Essential Oils Extracted from Lippia alba and Lippia citriodora

Indeed, anxiety is one of the most prevalent psychological problems during the pandemic4. According to a research, the prescriptions of antianxiety and sleep aid medication increased for 35.7% and 41.2% of the population respectively, because of the pandemic5. It is known that benzodiazepines (BDZs) and antidepressants are widely prescribed for anxiety disorders, nevertheless these drugs can produce side effects as lethargy, retrograde amnesia, drowsiness, dizziness, and vertigo6, as well to cause physical dependence and tolerance7. Besides, the abrupt cessation of BZDs can cause life-threatening seizures, delirium, and death8.


INTRODUCTION
COVID-19 pandemic has caused an unprecedented situation around the world, and one of the most significant consequence is the growing increase of mental health problems 1 , even the term coronaphobia was coined to describe the fear and anxiety caused by pandemic 2 , associated to high levels of depression, hopelessness, suicidal ideation, and functional impairment 3 .
Indeed, anxiety is one of the most prevalent psychological problems during the pandemic 4 . According to a research, the prescriptions of antianxiety and sleep aid medication increased for 35.7% and 41.2% of the population respectively, because of the pandemic 5 . It is known that benzodiazepines (BDZs) and antidepressants are widely prescribed for anxiety disorders, nevertheless these drugs can produce side effects as lethargy, retrograde amnesia, drowsiness, dizziness, and vertigo 6 , as well to cause physical dependence and tolerance 7 . Besides, the abrupt cessation of BZDs can cause life-threatening seizures, delirium, and death 8 .
Besides, there is a growing acceptation for Complementary and Alternative Medicine (CAM) treatments, since are perceived as natural, lowrisk, economical, with fewer side effects and as effective as conventional drugs 9 . In this context, aromatherapy employs essential oils (EOs), secondary metabolites mainly extracted from leaves, flowers, rhizomes, seeds, barks, and resins of aromatic plants 10 .
EOs are isolated via steam distillation, hydrodistillation, solvent extraction, enfleurage, mechanical pressing, carbon dioxide (CO 2 ) and supercritical CO 2 extraction, among others 11 . In aromatherapy EOs are applicated trough inhalation, massage, compress, and baths; in addition to being used as therapeutic agents and aesthetics 12 .
Evidence suggests that EOs could alleviate anxiety significantly showing short terms benefits 13 . Besides, a metanalysis found that aromatherapy significantly decreased pain and anxiety in the first stage of labor in nulliparous women 14 . In this order of ideas, a study examined the effects of rosa damascene oil on anxiety and sleep quality in cardiac patients, where this aroma significantly reduced anxiety in the experimental group compared to the control group 15 . In addition, an investigation found that aromatherapy based on EOs is a cost-effective method that can reduce anxiety in hospitalized patients 16 .
Lippia alba and Lippia citriodora are two aromatic shrubs that belong to the Verbenaceae family and have a wide distribution, especially in tropical and subtropical as well as temperate zones of the Americas, Africa, and Asia 17 . Traditionally, these species are consumed as tea or essential oil in Latin America and Africa due their sedative and tranquilizer properties 18 .
Despite that Lippia species are associated to an anxiolytic effect in traditional medicine and animal models 19,20 , there is lack of evidence about EOs in human models. In this sense, the objective of this study was to determine the anxiolytic-like effect of essential oils extracted from Lippia alba and Lippia citriodora in human volunteers.

Plant material
The leaves of Lippia alba and Lippia citriodora were collected from Cajamarca, a city in northern highlands of Peru at 2750 masl. The sample collection was conducted in the months of January to February 2021. Voucher specimens were prepared and identified by Segundo Leiva Gonzales, Biol, and deposited at the Herbarium Antenor Orrego (HAO) of Antenor Orrego University.

Essential oils extraction
The freshly collected leaves were washed with distilled water to remove dust. Then, samples were dried using a forced air circulation stove at a temperature of 40°C for 24 h. Next, leaves were milled, and the powdered plant material (100 g) were placed in a round bottom flask with 1000 ml distilled water, and then connected to a Clevenger-type apparatus. Hydrodistillation was completed for 3 h after boiling. Then the oil was dried over anhydrous sodium sulphate and stored in a refrigerator in amber glass vials at 4°C for further use in experiments 21 . The same procedure was carried out for both species.

Study design and sample
An experimental study with measures at pretest-posttest was conducted, using two experimental groups and a waiting-list control group. 95 participants were divided into three groups of 32 participants for each experimental group, the first one treated with aromatherapy based on L. alba essential oil (EG1) y the second group treated with L. citriodora essential oil (EG2); and finally, a wait-list (WL) control group with 31 participants.

Study procedure
A free aromatherapy course was offered through social media to recruit participants. The course was conducted entirely online, due to government restrictions due to the global pandemic. 104 people were enrolled and 95 took part in this investigation between April and May 2021. Inclusion criteria included participants men and female between the ages of 18-55 and they were required to have a State-Trait Anxiety Inventory score of greater than 20 in both scales; meanwhile exclusion criteria were participants with previous practice of alternative therapies such as meditation, tai chi or yoga, psychiatric or psychological treatment, and pregnancy. Participants for each group were randomized by a person not involved in the study by utilization of a random number table. After groups were formed, instruments were administered (pretest). Besides, each participant received weekly an aromatherapy kit with everything they needed for applications, including an instruction manual. Each kit was sent each week to each participant until the investigation was completed. In addition, weekly online meetings were held to monitor the applications and provide feedback. The EOs applications were according to the methodology of Reza, Bazeli, Basiri and Aalami 23 , where the participants were asked to pour two drops of EOs on a cotton ball using a dropper. Next, the cotton ball was held under the participant's nose while closed his or her eyes and took 10 deep breaths. Then, the cotton was pinned to the collar of the participant for 30 minutes. After that, the participant unpinned and disposed the cotton ball. WL control group did not receive any intervention until applications in EG1 and EG2 ended. The applications were every day for 4 weeks. At the end of interventions, an anxiety self-report instrument was administered (posttest). (Fig. 1). When the offered course finished, all participants were informed about the investigation program goals and signed a consent form in which confidentiality and anonymity were guaranteed. The study protocol was approved by Institutional Review Board (IRB) of Universidad Nacional de Trujillo. Besides, this investigation was performed in accordance with the Declaration of Helsinki.

Instruments
Anxiety was evaluated using the State-Trait Anxiety Inventory "STAI", which consists of two self-report scales measuring two distinct types of anxiety: state (actual levels of intensity and anxiety states) and trait (selects individuals who vary in their tendency to react to psychological stress with varying degrees of intensity). Both scales consist of 20 statements and respondents rate the intensity of their feelings about each at that moment from 1 (not at all) to 4 (very much so). The part that regards trait describes how the subjects generally feel, while the part that regards state describes how they feel at a given moment 12,24 . Inventory was validated for local population in a previous study 25 .

RESULTS
Besides, Table 4 shows the scores obtained by the Cohen's D test, Hedges' G and the percentage of change found; where values between 0.5 and 0.8 show moderate changes, as is the case of trait anxiety in EG1(d = 0.66; g = 0.66) and state (d = 0.78; g = 0.78) and trait (d = 0.49; g = 0.48) anxiety in EG2, while values higher than 0.80 show a large' effect size as evidenced for state anxiety in EG1 (d = 0.93; g = 0.93). Likewise, the percentages of change showed that state and trait anxiety in EG1 obtained a percentage of change of -14.78%, and -13.18% respectively, in addition, state and trait anxiety in EG2 obtained a percentage of change of -11.67%, and -10.51% respectively.

DISCUSSION
EOs are metabolites of plants that are typically oily liquids at ambient temperatures. In this case, chemical composition showed that L. alba essential oil can be classified as carvone chemotype due to this component is the main according to outcomes. This agrees with data from other investigations where it is also reported this chemotype for Peru 26,27 . However, L. citriodora essential oil showed that geranial, neral and limonene were the compounds where the major quantitative differences were observed. Although it is true that there are no studies on the chemical composition of the essential oil of this species in Peru, this coincides with other studies where they also show these two components as the main ones in the composition of this essential oil 28,29 .
Regarding socio-demographic data of participants, the majority were adults with graduate and postgraduate level of education. This is in concordance with some studies which affirm that most educated population is more likely to be interested in complementary and alternative medicine therapies like aromatherapy 30 . Nevertheless, this point constitutes one of the limitations because only few participants belonged to least educated population and our study does not show how these therapies can work in a different population, besides, the participant number is insufficient hence results cannot be generalized.
Literature describes that EOs can regulate brain health and functions associated with mood, anxiety and depression 31 . Indeed, evidence in anxiety animal models and some clinical trials show that EOs used in aromatherapy can alleviate anxiety symptoms 10 . This data agrees with our findings where participants in EG1 and EG2 show a decrease in anxiety scores after intervention. Although it is true that anxiety levels are still high according to STAI, the decrease in anxiety scores generates a certain effect size denoting the intervention effectivity. In this sense, state anxiety scores showed a large size effect in EG1, however it was found medium size effect in outcomes belonging to the rest of categories in both experimental groups. This is in accordance with other studies which investigated the effectiveness of EOs in reducing state anxiety 32 .
A meta-analysis indicated that aromatherapy could mitigate especially temporary anxiety 13 . It is also remarkable to affirm that state anxiety is related to temporary situations that changes every moment and when disappear, the individual no longer experience anxiety, but trait anxiety is related to permanent personality features of people 33 .
Moreover, significant differences between experimental groups may be due to the fact of difference constitution of the two essential oils tested. In view of this, L. alba essential oil shows the major quantitative differences in components with anxiolytic effect in comparison with L.    citriodora. Indeed, carvone is considered as a depressor of the central nervous system, interacting with GABA A receptors in the brain after crossing the blood-brain barrier 34 . The mechanism is similar to BDZs that exert a positive allosteric modulatory effect increasing inhibitory GABAergic neurotransmission 35 . Furthermore, the highest amount of linalool was found in L. alba compared to L. citriodora. The evidence about anxiolytic effect of linalool inhalation is widely reported, although its anxiolytic mechanism of action is not clearly, studies suggest that anxiolytic effects are triggered by olfactory input evoked by linalool odor exposure that activate anxiolytic circuits involving GABAergic transmission via BDZ-responsive GABA A R 36 .
Limonene is another component present in both EOs, in this case, a study demonstrate that this component can inhibit anxiety-related behavior through A2A receptor-mediated regulation of DAergic and GABAergic neuronal activity 37 . Besides it is possible that terpenes such as geranial and neral present in L. citriodora, also exert anxiolytic action 38 . However, not only EOs components by themselves may present anxiolytic effects, but there is also evidence that properties of EOs cannot be attributed to a single component because these could be the result of the synergistic effects of its constituents 39 .

CONCLUSION
According to our investigation, the essential oils extracted from Lippia alba and Lippia citriodora may be useful as a mean to counteract anxiety, specifically state anxiety showing short-terms benefits.