Relaxing Effects of Breathing Pseudotsuga menziesii and Lavandula angustifolia Essential Oils on Psychophysiological Status in Older Adults

We evaluated the effects of breathing Pseudotsuga menziesii (P. menziesii) and Lavandula angustifolia (L. angustifolia) essential oils (EOs) during a horticultural activity on older adults. A total number of 92 older adult (71.2 ± 7.7 years old) participants were guided through a leaf printing procedure. In the meantime, water vapor and EOs were diffused in an orderly manner. The heart rate variability-related parameters as well as the brain waves were recorded. In addition, we also collected data for the State–Trait Anxiety Inventory-State (STAI-S) questionnaires before and after the whole indoor natural activity program. The physiological parameters including standard deviation of normal to normal intervals, normalized high frequency (nHF), and high alpha wave increased while the normalized low frequency (nLF), the ratio of LF-to-HF power, high beta wave, and gamma wave decreased following the breathing of P. menziesii and L. angustifolia EOs. These changes indicated a relaxing effect of breathing both EOs during a horticultural activity on older adults. Our results demonstrated a beneficial effect of P. menziesii EO which is as good as a well-known relaxant L. angustifolia EO. This notion was supported by the results of STAI-S. Here we developed an indoor natural activity program for older adults to promote physical and mental health.


Introduction
In modern urbanized society, encounters with nature are valuable and stress-relieving experiences for citizens. Nature-contacting activities such as forest recreation and horticultural experiences are believed to be beneficial to health. For example, recent studies reported that exposure to nature for at least 120 min per week is associated with good health and wellbeing [1,2]. Notably, even as short as 3.5 h of forest walking improves the physical and mental health of middle-aged and older adults [3]. Our group also reported the beneficial effects of various horticultural activities on adult populations [4].
Humans have lived in natural environments for most of their evolution. Therefore, human physiological functions are best suited to the natural environment, which is why the natural environment can enhance human psychophysiological health [5]. Park et al. [6] conducted experiments in 24 different forest environments in Japan. Through the analysis of many research data, the results prove that compared with the urban environment, the forest environment can reduce the concentration of cortisol, pulse rate, and blood pressure; increase parasympathetic nerve activity; and decrease sympathetic nerve activity, allowing the human body to feel relaxed on a physiological level. It also promotes positive emotions, enhances comfort, and lifts spirits.
A study by Li et al. [7] in Japan pointed out that after three days and two nights of forest bathing, the activity of human natural killer cells (NK) was enhanced, along with the number of NK cells and anti-cancer proteins in lymphocytes, and this effect persisted tion or a clinical history of epilepsy, asthma, or allergy to EOs were excluded. After fully understanding the purpose and procedures of the study, all participants were asked to sign the study participant consent form voluntarily. This study was approved by the Research Ethics Committee of National Taiwan University (NTU-REC No. 202104HM009).

Essential Oils
P. menziesii and L. angustifolia EOs as well as the EO diffusers were purchased from dōTERRA International LLC (Pleasant Grove, UT, USA). The diffusers were used to generate water vapor with or without EO in the experimental environment. The concentration of EO in the diffusor was 2.5% (0.5 mL EO in 20 mL water) as suggested [40]. The major chemical components of each EO provided by the producer are listed in Table 1.

Indoor Horticultural Activity
We chose leaf printing as an indoor horticultural activity for the older adult participants in our study. The leaf printing procedure consists of printing the colors of leaf veins or petals onto cotton fabric by pressing a marble ball [4]. A thin plastic sheet was placed between the leaves or flower petals and the marble ball to reduce the exposure of scent while pressing the ball. The participants were instructed and guided to finish their artwork by experienced experimenters in 60 min.

Experiment Procedure
This study was conducted at the Department of Horticulture and Landscape Architecture, National Taiwan University, between 10:00 a.m. and 4:40 p.m., from 4 November 2021 to 4 January 2022. To ensure the quality of the experiment, the number of participants in each group was limited to 16. To reduce the confounding factors, caffeinated or alcoholic beverages were prohibited; metal objects such as necklaces, watches, earrings, and rings were removed during the whole session.
In this study, we aimed to evaluate the effects of breathing EO during an indoor horticultural activity by monitoring the physiological parameters and collecting questionnaires before and after the whole session. The procedure of the experiment contained seven time periods (Figure 1). During the first period (10 min), participants were guided through the experimental environment and completed the informed consent form of the study. During the second period (15 min), participants were asked to wear patch-type sensors. The HRV and brain waves were continually collected as the baseline data. All participants also finished a State-Trait Anxiety Inventory-State (STAI-S) questionnaire during this period as the pre-test emotional status for reference. In the following period (15 min), an indoor horticultural activity, leaf printing, was introduced to the participants. In the meantime, water vapor without EO was diffused into the air. HRV and brain waves were also continually collected until the end of the experiment. During the next period (15 min), water vapor with P. menziesii EO was given via the diffuser. The EO could be inhaled by the participants via normal breathing. During the following 15 min period, between the first and second EOs, water vapor without EO was diffused. Finally, L. angustifolia EO was given for another 15 min. At the end of the test, the leaf printing activity also finished and the physiological recordings were then ceased and detached. All participants were asked to complete another STAI-S questionnaire which was used to reflect the post-test emotional status.
sensors. The HRV and brain waves were continually collected as the baseline data. All participants also finished a State-Trait Anxiety Inventory-State (STAI-S) questionnaire during this period as the pre-test emotional status for reference. In the following period (15 min), an indoor horticultural activity, leaf printing, was introduced to the participants. In the meantime, water vapor without EO was diffused into the air. HRV and brain waves were also continually collected until the end of the experiment. During the next period (15 min), water vapor with P. menziesii EO was given via the diffuser. The EO could be inhaled by the participants via normal breathing. During the following 15 min period, between the first and second EOs, water vapor without EO was diffused. Finally, L. angustifolia EO was given for another 15 min. At the end of the test, the leaf printing activity also finished and the physiological recordings were then ceased and detached. All participants were asked to complete another STAI-S questionnaire which was used to reflect the post-test emotional status.

Physiological Recordings and Analyses
Throughout the experimental period, wearable patch-type sensors for HRV and EEG (BeneGear Inc., New Taipei City, Taiwan) were attached to the chest and forehead, respectively. The HRV and EEG data were continuously stored in the sensors during recording and then transferred to a computer for further processing and analysis.

Emotional Evaluation
The State-Trait Anxiety Inventory (STAI) questionnaire includes 40 self-assessment questions, 20 for state anxiety (STAI-S) and 20 for trait anxiety (STAI-T), respectively [41]. The STAI-S is used to assess anxiety levels at a specific time and as an indicator in certain special stressful situations. The STAI-T measures the general feeling of an individual's During the whole experiment, the indoor air was monitored using an AirBoxx air quality meter (KD Engineering, Glen Cove, NY, USA). The CO 2 concentration, CO concentration, temperature, and relative humidity in the experimental environment were 889.8 ± 337.7 ppm, 0.18 ± 0.4 ppm, 22.6 ± 2.1 • C, and 74.9 ± 6.4%, respectively.

Physiological Recordings and Analyses
Throughout the experimental period, wearable patch-type sensors for HRV and EEG (BeneGear Inc., New Taipei City, Taiwan) were attached to the chest and forehead, respectively. The HRV and EEG data were continuously stored in the sensors during recording and then transferred to a computer for further processing and analysis.

Emotional Evaluation
The State-Trait Anxiety Inventory (STAI) questionnaire includes 40 self-assessment questions, 20 for state anxiety (STAI-S) and 20 for trait anxiety (STAI-T), respectively [41]. The STAI-S is used to assess anxiety levels at a specific time and as an indicator in certain special stressful situations. The STAI-T measures the general feeling of an individual's anxiety proclivity. In the present study, we used an STAI-S, comprising 20 questions, including ten positive and ten negative items. Each item is scored using the four-point Likert scale, 1 to 4 for negative questions and 4 to 1 for positive questions, respectively. The higher scores indicate higher anxiety levels [42,43].

Statistics
IBM SPSS Statistics for Windows, Version 21.0, was used to analyze all data (IBM Corporation, Armonk, NY, USA). Except for the HR, the sex-dependent differences in all physiological parameters and emotional scores were insignificant; we, therefore, pooled data collected from both genders together in each test and question. Data are expressed as the mean ± standard error (Mean ± SE). Non-parameter statistics: the Wilcoxon signedrank test was used to compare the differences between baseline and treatment (water or EOs). A p-value < 0.05 was considered statistically significant.

Physiological Recordings and Analyses
To evaluate the effects of breathing EOs during an indoor horticultural activity, leaf printing, a wearable HRV sensor was used to continuously record HR-related parameters. During the leaf printing procedure, water vapor, water vapor with P. menziesii EO, water vapor, and water vapor with L. angustifolia EO were diffused into the air in an orderly manner. The baseline activity was recorded before the beginning of the horticultural activity. The HRs of the participants were not significantly altered during the whole experiment. However, HRV parameters were affected by the breathing of EOs. Compared with the baseline, the value of the standard deviation of normal to normal intervals (SDNN) was not changed while water vapor was diffused during the procedure of leaf printing; notably, SDNN was increased while P. menziesii EO and L. angustifolia EO were given ( Figure 2A). SDNN represents the intensity of autonomic regulation of the sinus node and thus the overall activity of the autonomic nervous system, and is an indicator of physiological resilience to stress [44][45][46][47][48]. We next analyzed the percentages of normalized low frequency (nLF) and high frequency (nHF) that reflect the sympathetic and parasympathetic activity, respectively [22,24,49]. Diffusion of water vapor did not affect nLF and nHF, however, the percentages of nLF decreased while P. menziesii EO and L. angustifolia EO were diffused ( Figure 2B). Correspondingly, the percentages of nHF increased when the participants were breathing both EOs ( Figure 2C). The ratio of nLF to nHF (LF/HF) indicates the autonomic activity balance [23,44]. We also observed decreased LF/HF when both EOs were given whereas water vapor alone did not affect LF/HF ( Figure 2D). Together, these results demonstrated a physiological condition of decreased sympathetic and increased parasympathetic activities, indicating a relaxing status during breathing of EOs.
A wearable EEG sensor was also used to continuously record brain wave-related parameters. The baseline activity was recorded before the beginning of the horticultural activity. We analyzed the high alpha (10-13 Hz) wave, which is frequently represented as a relaxed but focused brain wave [29,50,51]. Compared with the baseline, the value of the high alpha wave was increased significantly during the exposures of both P. menziesii EO and L. angustifolia EO. The diffusion of water vapor without EO did not alter the high alpha wave ( Figure 3A). Further, we analyzed the high beta (20-30 Hz) wave, which is commonly associated with stressful situations [52,53]. The high beta wave was reduced while breathing both P. menziesii EO and L. angustifolia EO (Figure 3B), indicating a reduction in stress level. Interestingly, the value of the high-beta wave in the second water vapor section was significantly lower than the baseline level, suggesting a long-lasting effect of P. menziesii EO. We also analyzed the feature of the gamma (30-100 Hz) wave, which is negatively associated with the level of relaxation [54][55][56][57][58]. The gamma waves reduced while the participants were breathing EOs (Figure 3C), indicating a more relaxed condition. Together, these EEG data also suggested an EO-dependent relaxing effect and reduction in stress levels.  A wearable EEG sensor was also used to continuously record brain wave-related parameters. The baseline activity was recorded before the beginning of the horticultural activity. We analyzed the high alpha (10-13 Hz) wave, which is frequently represented as a relaxed but focused brain wave [29,50,51]. Compared with the baseline, the value of the high alpha wave was increased significantly during the exposures of both P. menziesii EO and L. angustifolia EO. The diffusion of water vapor without EO did not alter the high alpha wave ( Figure 3A). Further, we analyzed the high beta (20-30 Hz) wave, which is commonly associated with stressful situations [52,53]. The high beta wave was reduced while breathing both P. menziesii EO and L. angustifolia EO (Figure 3B), indicating a reduction in stress level. Interestingly, the value of the high-beta wave in the second water vapor section was significantly lower than the baseline level, suggesting a long-lasting effect of P. menziesii EO. We also analyzed the feature of the gamma (30-100 Hz) wave, which is negatively associated with the level of relaxation [54][55][56][57][58]. The gamma waves reduced while the participants were breathing EOs ( Figure 3C), indicating a more relaxed condition. Together, these EEG data also suggested an EO-dependent relaxing effect and reduction in stress levels.  . The brain-waves-related physiological parameters. After the baseline period, an indoor horticultural activity, leaf printing, was introduced. In the meantime, water vapor (water), water vapor with Pseudotsuga menziesii essential oil (P.m. EO), water vapor (water), and water vapor with Lavandula angustifolia essential oil (L.a. EO) were diffused into the air in an orderly manner. The values of high alpha wave increased during the diffusion of both EOs (A). The values of high beta wave decreased while EOs were given (B). The gamma wave was reduced while breathing EOs (C). Data were collected from 92 participants and presented as mean ± SE. *, p < 0.05; **, p < 0.01, Wilcoxon signed-rank test.

Emotional Evaluation
The emotional status of the participants was evaluated using STAI-S questionnaires. The first questionnaire was given to the participants before the horticultural activity and diffusion of EOs as the pre-test emotional status. A second questionnaire was given after the completion of the experiment to evaluate the post-test emotional status. Compared Figure 3. The brain-waves-related physiological parameters. After the baseline period, an indoor horticultural activity, leaf printing, was introduced. In the meantime, water vapor (water), water vapor with Pseudotsuga menziesii essential oil (P.m. EO), water vapor (water), and water vapor with Lavandula angustifolia essential oil (L.a. EO) were diffused into the air in an orderly manner. The values of high alpha wave increased during the diffusion of both EOs (A). The values of high beta wave decreased while EOs were given (B). The gamma wave was reduced while breathing EOs (C). Data were collected from 92 participants and presented as mean ± SE. *, p < 0.05; **, p < 0.01, Wilcoxon signed-rank test.

Emotional Evaluation
The emotional status of the participants was evaluated using STAI-S questionnaires. The first questionnaire was given to the participants before the horticultural activity and diffusion of EOs as the pre-test emotional status. A second questionnaire was given after the completion of the experiment to evaluate the post-test emotional status. Compared with the pre-test scores, the STAI-S scores of the post-test decreased significantly in all questions (Figure 4). In the scoring system, higher scores denote higher anxiety levels [42,43]. Our findings indicated that the anxiety level of the participants was reduced after the horticultural activity and breathing of EOs.

Discussion
This study established a procedure to analyze physiological parameters including HRV and brain waves and evaluate the relaxing effects of breathing P. menziesii and L. angustifolia EOs during a horticultural activity on older adults.
Encounters with nature, such as forest bathing, are healthful. People in natural environments exhibit lower sympathetic activity than in urban environments and increased parasympathetic activity [6,9,59,60]. However, residents in modern society have a hectic pace of life, and time is often limited; forest recreation is usually difficult to achieve. Programs that incorporate elements of the forest environment such as natural light, plant- . STAI-S scores before and after the horticultural activity and diffusion of EOs. In all questions, the scores decreased dramatically in the post-test STAI-S questionnaire. @ indicates a positive question. Data were collected from 92 participants and presented as mean ± SE. *, p < 0.05; ***, p < 0.001, Wilcoxon signed-rank test.

Discussion
This study established a procedure to analyze physiological parameters including HRV and brain waves and evaluate the relaxing effects of breathing P. menziesii and L. angustifolia EOs during a horticultural activity on older adults.
Encounters with nature, such as forest bathing, are healthful. People in natural environments exhibit lower sympathetic activity than in urban environments and increased parasympathetic activity [6,9,59,60]. However, residents in modern society have a hectic pace of life, and time is often limited; forest recreation is usually difficult to achieve. Programs that incorporate elements of the forest environment such as natural light, plant-based features, organic textures, sounds, and aromas to integrate nature into human life, are being developed to promote mental and physical health [59,61]. For example, stress-relieving effects have been shown during indoor horticultural activities [4]. Here, we combined indoor horticultural activities with the breathing of P. menziesii and L. angustifolia EOs as an indoor natural activity program and aimed to create an indoor natural environment where the participants are immersed in natural elements close to forest bathing.
We analyzed the physiological status of the participants including HRV and brain waves during the indoor natural activity. HRV could be used to evaluate the activity of ANS including sympathetic and parasympathetic divisions. SDNN represents the overall activity of the ANS and denotes an indicator of physiological resilience to stress [44][45][46][47][48]; while the percentages of nLF and nHF reflect the sympathetic and parasympathetic activity, respectively [22,24,49]. Brain waves, on the other hand, could be used to evaluate mental status. The power of alpha waves increases when people are relaxed and at rest [29,[62][63][64].
While the values of high beta positively correlated with the stress level [27,63,64], the gamma wave is negatively associated with the level of relaxation [54][55][56].
Breathing of EOs has benefits for mental and physiological status [65]. Our physiological results, including increased SDNN, nHF, and high alpha wave activity accompanied by reduced nLF, LF/HF, and high beta wave and gamma wave during the exposure of L. angustifolia EO, indicated a relaxation effect. Our findings are in line with previous reports [24,[66][67][68][69]. The main component of L. angustifolia EO is linalool (36.03%), which has relaxation and anxiolytic functions [70,71]. Further, its cognitive-enhancing effects such as a decrease in arousal level and an improvement in the sustained component of attention has also been proposed [72]. The benefits of breathing L. angustifolia EO are evident.
Compared with the findings during breathing L. angustifolia EO, similar physiological results were obtained while defusing P. menziesii EO, indicating a relaxation effect. To the best of our knowledge, this is the first trial of using physiological approaches to verify the function of inhaling P. menziesii EO. The main components of P. menziesii EO include β-pinene (21.16%) and α-pinene (14.97%). Pinenes are monoterpenes that have various functions including antimicrobial activity [73] as well as anti-inflammatory, antioxidant, and neuroprotective properties [71]. Further, the anxiolytic effect of pinenes has been proposed [74][75][76][77][78]. Inhaled pinenes may modulate the serotonergic, adrenergic, and dopaminergic systems in the brain as well as the expression of BDNF in the hippocampus that are involved in the pathogenesis of depression and anxiety [71]. Based on the physiological findings, our results demonstrated a relaxation effect of P. menziesii EO; the pinenes could be the active component for this function.
The use of the STAI questionnaires could provide more insight into the subjective psychological state of the participants [42,43,54,60,79,80]. We evaluated the anxiety scores of the participants by collecting STAI-S questionnaires before and after the whole program. As for the results of the STAI-S questionnaires, the scores all decreased after the indoor natural activity program, indicating a stress-relieving effect. This is consistent with the previous findings using STAI-S that the aroma of EOs can reduce anxiety in dental patients [37,39] primiparous women during childbirth [81], and university students [82].
In our present study, P. menziesii and L. angustifolia EOs were diffused during the horticultural activity. The effects of EO were prominent because the changes in HRVrelated parameters and brain waves during EO diffusion diminished when water vapor was diffused. Our goal was to compare the HRV and brain waves in different conditions of the same individuals. In this design, the diffused water vapor was used as the control to avoid affecting the accuracy of relaxation benefits due to differences in the innate physical and mental adjustment ability between individuals. The 15 min duration for water and EO was chosen based on the short-term adaptation property of the olfactory system [83][84][85][86].

Limitations
There are limitations in this study. A total number of 92 participants (11 males and 81 females) was included. The results were mostly collected from females (88%). Although the sex-dependent differences in all physiological parameters and emotional scores were insignificant in this study, we still need to recruit more male participants for comparison. In addition, the wearable EEG device was attached to the forehead in this study and so the brain waves were mostly recorded from the prefrontal cortex. The neural activities in other brain areas were not recorded and analyzed.

Future Research
Similar to linalool, a major component of L. angustifolia EO, the cognitive-enhancing effects of pinenes, and major components of P. menziesii EO were demonstrated [71]. However, these findings were collected from mice [87] and fruit flies [88]. It is worth testing if P. menziesii EO improves learning and memory in humans, especially older adults. Since the sense of smell plays a significant role in the biopsychological effects of stress, mood, and work capacity [20,30,38,89,90], diffusion of P. menziesii and L. angustifolia EOs could also be suitable for people of other ages. We should extend the study to other age groups in the future.

Conclusions
In this study, we developed a natural element-based program combining indoor horticultural activity and diffusion of P. menziesii and L. angustifolia EOs and analyzed the psychophysiological status of the participants. This indoor natural activity program has relaxation and stress-relieving effects and is believed to benefit the health of older adults.  Data Availability Statement: The data will be made available through the request to the corresponding author.