The effects of aromatherapy essential oil inhalation on stress, sleep quality and immunity in healthy adults: Randomized controlled trial

Accepted Manuscript Title: The effects of aromatherapy essential oil inhalation on stress, sleep quality and immunity in healthy adults: randomized controlled trial Authors: Mi-kyoung Lee, Sunog Lim, Ji-Ah Song, Mi-Eun Kim, Myung-Haeng Hur PII: DOI: Reference:

S1876-3820(17)30095-1 http://dx.doi.org/doi:10.1016/j.eujim.2017.04.009 EUJIM 671

To appear in: Received date: Revised date: Accepted date:

17-3-2017 20-4-2017 24-4-2017

Please cite this article as: Lee Mi-kyoung, Lim Sunog, Song Ji-Ah, Kim Mi-Eun, Hur Myung-Haeng.The effects of aromatherapy essential oil inhalation on stress, sleep quality and immunity in healthy adults: randomized controlled trial.European Journal of Integrative Medicine http://dx.doi.org/10.1016/j.eujim.2017.04.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The effects of aromatherapy essential oil inhalation on stress, sleep quality and immunity in healthy adults: randomized controlled trial

Mi-kyoung Lee1*, Sunog Lim2*, Ji-Ah Song3, Mi-Eun Kim4, Myung-Haeng Hur1 1

College of Nursing, Eulji University,2Department of Nursing Science, Shingyeong University,

3

Department of Nursing, Woosong College,4Department of Nursing, Gimcheon University,

* EqualContributor Corresponding Author: Myung-HaengHur College of Nursing, Eulji University, Daejeon, Korea Address : 77 Gyeryong-ro 771 beon-gil, Jung-gu, Daejeon 34824, Korea TEL : +82-42-259-1714, FAX : +82-42-259-1709 E-Mail :[email protected], [email protected], [email protected] Mi-kyoung Lee

Sunog Lim

Ji-Ah Song

Mi-Eun Kim

MyungHaengHur

College of Nursing, Eulji University Address : 77 Gyeryong-ro 771 beon-gil, Jung-gu, Daejeon 34824, Korea TEL : +82-42-259-1712, FAX : +82-42-259-1709 Department of Nursing Science, Shingyeong University Namyang-ro 400, Hwaseong-si, Gyeonggi-do 445-741 TEL: +82-10-8849-7735, FAX: +82-31-369-9116 Department of Nursing, Woosong College Address : 171, Dongdaejeon-ro, Dong-gu, Daejeon, 34606, Korea TEL : +82-42-629-6746, FAX : +82-42-629-6749 Department of Nursing, Gimcheon University, Gimcheon, Korea TEL : +82-54-420-4227 College of Nursing, Eulji University Address : 77 Gyeryong-ro 771 beon-gil, Jung-gu, Daejeon 34824, Korea TEL : +82-42-259-1714, FAX : +82-42-259-1709

1

E-Mail : [email protected]

E-Mail : [email protected]

E-Mail : [email protected]

E-Mail : [email protected]

E-Mail : [email protected], [email protected], [email protected]

Abstract Introduction: Stress can lead to poor sleep and compromise immune function and it is important to identify approaches that can address such problems and improve quality of life. The aim of this randomized controlled trial was to examine whether aromatherapy via inhalation of essential oils could

relieve perceived stress, depression, sleep quality, and

immune function. Methods: Subjects aged 20–60 years responding to a recruitment advertisement posted in a general hospital were randomly assigned into either an aromatherapy group (n = 30) or a waiting list control group (n = 30). The experimental treatment was to inhale an essential oil blend of lemon, eucalyptus, tea tree, and peppermint in a ratio of 4:2:2:1. The essential oil blend was inhaled by wearing a pendant during the day and sleeping near an aromatherapy stone at night for four weeks. Perceived stress, stress index, autonomic nervous system (ANS) activation, and glycated hemoglobin (HbA1c) levels were measured to examine stress. In addition, depression was measured using the Center for Epidemiologic Studies Depression Scale (CES-D). Sleep quality and immune state were also measured. Results: The aromatherapy group had significantly lower perceived stress levels (p<.001) and depression (p=.049) and significantly higher sleep quality(p=.001), but the two groups did not differ in ANS activation, HbA1c levels, or immunity. Conclusions: In conclusion, inhalation of essential oils as per aromatherapy, resulted in lower perceived stress and depression, as well as better sleep quality, but did not influence physiological parameters, such as the stress index or immune state. Key Words: Aromatherapy, Stress, Depression, Sleep, Immunity, Essential Oil, randomized controlled trial 2

1. Introduction Infectious disease epidemics, such as herpes, herpes zoster, cold, influenza, and swine flu, as well as the increasing prevalence of chronic diseases, such as cancer have been associated with an increase in stress and reduced immunity to infection [1,2].

This has in

turn has inspired vigorous research on the relationship between stress and sleep, and the role of stress, infection, and immunity[3]. Several medical and non-medical measures to relieve stress, improve sleep quality, and boost immunity have been proposed[4, 5]. Stress relief, improvement of sleep quality, and improvement in immune function are key factors not only in prolonging a healthy lifespan, but also in increasing quality of life. Stress is a concept derived from physics and engineering, adopted by psychology and biology. It has been defined as an atypical physiological reaction that arises within the body in response to imagined or actual injury and various stimuli inflicted on the body[6]. Symptoms of stress may include reduced judgment, excessive worrying, instability, agitation, depression, pain, digestive symptoms, nausea, vertigo, chest pain, tachycardia, increased or reduced appetite, increased or decreased sleep, social withdrawal, drinking or smoking, and increased drug intake [6]. While there are many symptoms of stress as outlined above, depression is frequently associated with stress. Depression refers to “negative emotions,” and presents as an imbalance of neurotransmitters, such as dopamine, serotonin, and norepinephrine. Major symptoms of depression include lethargy, anxiety, sleep disturbances, loss of interest, suicide, and physical symptoms, such as headache and indigestion [7]. Sleep difficulties are also associated with stress and are often also related to depression. Maintaining quality of sleep is especially important to overall health, as reduced sleep caused 3

by stress is related to decrease immune function [8, 9]. During sleep, the body’s sensory activities and voluntary muscles are suppressed, and interaction with the environment is reduced. Sleep is divided into non-rapid eye movement (NREM) and rapid eye movement (REM) phases; a sleep cycle begins with NREM sleep and progresses to REM sleep, and is repeated 4–5 times a night in 90-minute intervals. NREM sleep is characterized by recovery of the body’s tissues, while REM sleep is characterized by increased protein synthesis and recovery of brain tissue functions, in addition to physical and psychological recovery [8, 10]. Various factors interrupt sleep, but depression has been specifically correlated with sleep disorders in many studies. For example, individuals with more severe insomnia have shown increased depression severity, demonstrating a positive correlation between sleep disorders and depression [11]. There is a cyclical relationship between these factors such that depression induces sleep disorders, and sleep disorders result in depression [12]. Therefore, ensuring an adequate amount of sleep and improving sleep quality could be one method to relieve stress and depression. Immune function is also closely related to stress and depression. Immunity refers to a host's resistance to non-self substances[13]. Several recent studies have suggested that emotional factors play a central role in immune function. For example, chronic stress particularly influences the brain, hormones, visceral fat, and immune system. Specifically, chronic stress reduces memory and increases stress hormones and visceral fat, which in turn makes the host vulnerable to various diseases, such as heart and infectious diseases. For example, it has been reported that social disruption causes changes in immune cell reaction to infection, resulting in respiratory inflammation [14]. Furthermore, depression not only decreases atypical immune responses, but also activates inflammatory processes; therefore, 4

herpes zoster is especially more risky and severe in older individuals and those with depression [15]. Stress has also been reported to heighten individual’s vulnerability to infectious diseases, as evidenced by an assessment of antibody responses to an influenza vaccine in a stressful situation [16]. Taken together, these findings suggest that stress is associated with immunity, a connection that has been proposed since the discovery of stress by Hans Selye[9]. In conclusion, developing antibiotics and devising measures to enhance immune function have become highly important due to various epidemic outbreaks and the rise in the prevalence of chronic diseases. There have been continuous advances in developing antistress measures, sleep quality improvements, medical and nursing interventions, including antimicrobial and immune-enhancing techniques, as well as the use of complementary and alternative medicine (CAM) treatments. Traditional treatment of infectious diseases typically involves antibiotics, and nursing interventions which can involve helping patients to overcome disease through comfort and rest. In addition, CAM to relieve stress has included: meditation, music therapy, art therapy, aromatherapy, Taichi, yoga, and laughter therapy. Aromatherapy uses essential extracts from plants, specifically, the use of herbs and their essential oils. Several essential oils used in aromatherapy have been suggested to have antimicrobial effects, preservative effects, anti-stress effects, anti-depressive effects, antiinflammatory effects, relaxation effects, and immune-enhancing effects[17]. Essential oils that are recommended for aromatherapy include tea tree, cinnamon, clove, eucalyptus, thyme, rosemary, lavender, pine, and tea tree oil, which is one of the more well-known immuneenhancers [17]. The recommended methods of using these essential oils are through bath, inhalation, and massage. 5

Literature pertaining to the anti-stress effects of aromatherapy is accumulating in Korea and abroad, and the evidence of anti-stress effects produced by CAM therapies have been adequately established[18]. However, there is still a lack of evidence supporting the theory that stress depresses immune function and decreases quality of sleep. Several studies have examined anti-stress effects of the use of aromatherapy essential oils [19-21], but such studies are still few in number, with a particular scarcity of research on immune-enhancing effects of essential oils. Despite the significance of immunity in inhibiting disease morbidity, it is difficult to measure immunity. In addition, although several parameters could be used to measure immunity, these are also difficult to study because they are influenced by various situational, psychological, and physical factors. Therefore, there is still a lack of research on the immune-enhancing effects of aromatherapy, which suggests there is a need for additional studies on this matter, as prevalence of various infectious diseases is on the rise and many diseases are caused by decreased immune function. Several essential oils used for aromatherapy have been suggested to have immune-enhancing effects, and researchers are beginning to verify these effects. For example, a study that examined the effects of lavender on citrobacterrodentium-induced enteritis found that lavender had immune-enhancing effects [22]. Moreover, another study examined the effects of aromatherapy provided through Thai massage using coconut oil containing ginger oil on colorectal cancer patients receiving chemotherapy; measurements of patients’ T-lymphocytes, and cluster of differentiation 4 (CD4), CD8, and CD4/CD8 counts revealed that aromatherapy was effective in enhancing cell mediated immunity [23]. The present study seeks to verify the stress relief and immune-enhancing effects of aromatherapy essential oil inhalation. Ultimately, the aim of this study was to investigate 6

whether the quality of life of individuals could be improved by relieving stress and enhancing immunity. 2. Methods 2.1 Study design The present study adopted a randomized controlled trial design to compare the effects of aromatherapy oil inhalation on adults’ stress, depression, sleep quality, and immunity (Figure 1). 2.2 Subjects selection and randomization 2.2.1 Selection and assignment of subjects Study candidates were adults who enrolled through a recruitment advertisement posted at a general hospital between September 2015 and October 2015. Participants were randomly assigned to either the aromatherapy group or the waiting list group. The inclusion/exclusion criteria were as follows: adults aged 20 to 60 years old, full capability of communicating, no auditory or olfactory problems, no psychiatric disorders, and not currently taking any medications associated with enhancing immune function. All participants provided written informed consent and the protocol was approved by the Institutional Review Board at E hospital (EMCIRB 15-83). In addition participants were informed that they could withdraw from the study at any time without any repercussion. 2.2.2 Sample size calculation and randomization Sample size was calculated using G*power by entering the significance level (α), statistical power (1-ß), and effect size (effect size). To compare the means of the two groups, a medium effect size was set at 0.80, level of significance was 0.05, and statistical power was 0.80, with two equivalent groups. The G*power calculation suggested a total sample of 52 7

participants, with 26 in each group. In consideration of a 15% withdrawal rate, a total of 60 individuals were enrolled, with 30 randomly assigned to each of the aromatherapy and waiting list groups. Subjects were randomly assigned using a computerized random generator. None of the participants withdrew during the study, resulting in 30 participants in the aromatherapy group and 30 in the waiting list group in the final analyses (Figure 2). 2.3

Outcome measures

2.3.1 Primary outcomes 2.3.1.1 Stress To measure stress, we examined perceived stress and physiological stress in three separate sessions: pre-treatment, second week of treatment, and fourth week of treatment. 2.3.1.1.1 Perceived stress Perceived stress was measured using the Numeric Rating Scale (NRS)[24, 25]. The NRS instructs participants to mark their subjective level of stress on a horizontal line from 0 (I currently have no stress at all) to 10 (I am currently under severe stress). Responses were converted to NRS score, and a higher score indicates higher stress. 2.3.1.1.2 Physiological stress Stress index The stress index represents the degree of stress indicated by autonomic nervous system (ANS) balance based on changes in heart rate. The Stress index was measured by a trained research assistant using the Canopy 9 professional 4.0 system (IEMBIO, USA) for 5minutes. The stress index ranges from 1–10, and a higher stress index indicates subjects have higher stress. ANS activation 8

ANS activation represents the quantified area of low and high frequencies, based on changes in heart rate measured for five minutes. The low frequency (LF) area represents sympathetic nervous activity, with a higher value indicating more active sympathetic nerves. The high frequency (HF) area represents parasympathetic nervous activity, with a higher value indicating more active parasympathetic nerves. Glycated hemoglobin (HbA1c) If an individual perceives stress, the hypothalamus secretes corticotropin releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) that is released from the pituitary stimulates the adrenal cortex to secrete cortisol, a stress hormone, which in turn increases blood sugar level [26]. Glycated hemoglobin is glucose bound to hemoglobin, and its level reflects average blood sugar control within the past three months; a normal HbA1c level is 4– 6%. Blood sampling was performed by a career nurse and samples were analyzed in a hospital laboratory. 2.3.1.2 Sleep quality Sleep quality was measured using the sleep quality numeric rating scale (NRS). Participants mark their subjective sleep quality within the past week on a horizontal line from 0 (extremely bad) to 10 (extremely good), which is then converted to a score, with a higher score indicating better sleep quality[24, 25]. 2.3.1.3 Immunity To measure immunity, we obtained 3 cc of blood from each individual in an ethylenediaminetetraacetic acid (EDTA) tube to measure CD4, CD8, and CD16+56 counts, and another 3 cc in a serum separator tube (SST) to measure immunoglobulin (IgG). The reference values for the test were 870–1700 mg/dl for IgG, 28.4–56.4% for CD4, 20.9–46.9% 9

for CD8, and 7.2–34.5% for CD16+56. Blood sampling was performed by a career nurse. The samples were sent to and analytical laboratory. 2.3.2 Secondary outcomes 2.3.2.1 Depression Depression was measured using the Korean version of the Center for Epidemiologic Studies Depression Scale (CES-D), which was originally developed by the National Institute of Mental Health and validated by Cho and Kim [27], after obtaining permission from the researcher. The Korean CES-D is made up of 20-items that are on a 4-point scale, with a total score ranging from 20–80 points. In community epidemiology studies, a score of 21 points or higher determines depression. The Cronbach’s α when the Korean CES-D was first developed was .91 in the general population[27], and those in the present study pre-treatment, second week of treatment, and fourth week of treatment were .883, .910, and .920, respectively. 2.4 Interventions The experimental treatment was to inhale an essential oil blend of lemon, eucalyptus, tea tree, and peppermint in a ratio of 4:2:2:1 for four weeks. The used aromatherapy essential oils were manufactured by Neumond(Germany), and were blended by researcher who was a certified aromatherapist. It is produced by Neumond company (website: * http://www.neumond.de/english/base_qualitaet.html) in Germany and sold in Korea 's best being (website is http://www.bestbeing.co.kr/goods/goods_list.php?cateCd=001). Lemon oil is known to strengthen the immune system, relieve insomnia, and enhance mood, while eucalyptus, tea tree, and peppermint oils are known to have excellent antimicrobial and antiinflammatory effects [28]. For natural inhalation, participants were required to wear a pendant filled with essential oil during the day and placed an aroma stone with three drops of 10

the blended oil within 30 cm of themselves during the night. Therefore, Aromatherapy Essential Oils are naturally blended with lemon, eucalyptus, tea tree and peppermint oil blended in a 4: 2: 2: 1 ratio during the day. At night, three droplets were dropped on the aroma stone, and the subject was placed at a distance of 30 cm to inhale. Therefore, aromatherapy was applied to the aromatherapy group for 24 hours a day for a total of 4 weeks. The waiting list group did not receive any treatment for four weeks. However, upon conclusion of the 4-week intervention, after all data were collected, individuals in the waiting list group were also given the same aroma blended oil, pendant, and aroma stone with instructions on inhalation. 2.5 Data collection procedure Data were collected from November 5, 2015 to December 13, 2015. The data collection procedures were as follows(Figure 2): 1. Participants were recruited by posting an ad on the bulletin board at a general hospital, and they were selected based on specific inclusion criteria. 2. Participants were numbered in the order of enrollment from 1–60 and randomly assigned to either the aromatherapy group or the waiting list group using a randomization program (www.randomizer.org). 3. Preliminary testing was done to obtain participants’ general characteristics, stress and depression levels, sleep quality, and immune state. The whole experiment was conducted by two trained research assistants and blood samples were taken by two career nurses capable of blood sampling. The collected blood was put into each blood collecting container for examination and put in a moving box and sent to the laboratory safely by the person in charge of transport. After preliminary data were collected, the aromatherapy group was instructed to 11

inhale the aroma blended oil for four weeks using a pendant and an aroma stone, while the waiting list group did not receive any treatment. Before the experiment, subjects were given aroma oil, necklace(including spoid), and aroma stone. And we sent a text message to their cell phones twice a day to make sure they didn't forget to inhale the aroma oil. Postexperimental tests to compare the groups were carried out at two weeks and four weeks from the start of the intervention; stress, depression, sleep quality, and immune function were examined. 2.6 Data analysis Data were analyzed using SPSS Statistics, version 23.0 (IBM Corp., Armonk, NY, USA). Analysis of participants’ general characteristics and group homogeneity testing for the dependent variables were conducted via t-tests and chi-square tests. In addition, to compare the groups on stress, depression, sleep quality, and immune function, t-tests, analysis of covariance (ANCOVA), and repeated measures analysis of variance (ANOVA) were conducted. The reliability of the depression scale was analyzed based on its Cronbach’s α value. 3. Results 3.1 Pre-homogeneity analysis 3.1.1. Intergroup homogeneity in baseline characteristics of subjects A total of 60 individuals were enrolled in this study with 30 in each of the aromatherapy and waiting list groups. Homogeneity testing was performed prior to the treatment (W0), and the results of the intergroup homogeneity analysis for general characteristics were as follows. Table 1 gives details on the demographic characteristics of the 2 groups. All of the participants were female. Mean ages of the aromatherapy and waiting list groups were 37.3 12

years and 35.8 years, respectively. Mean heights and weights of the aromatherapy and waiting list groups were 159.6 cm and 56.2 kg, and 159.2 cm and 54.4 kg, respectively. Approximately 66.7% and 60.0% of the aromatherapy and waiting list groups, respectively, were married, and 80.0% and 83.3% of the aromatherapy and waiting list groups, respectively, were college graduates. All of the participants claimed to have no immune function disorders, but four individuals in the aromatherapy group (13.3%) indicated that they had a sleep disorder. Intergroup homogeneity testing of the general characteristics prior to conducting the experimental treatment showed that there were no significant differences between the two groups in age, height, weight, marital status, educational level, type of work, monthly income, caffeine intake, degree of drinking, exercise, presence of immune disorder, or presence of sleep disorder (Table 1). 3.1.2 Intergroup homogeneity in dependent variables In the present study, stress was measured based on the subjective stress score from the NRS and physiological stress scales (i.e., Canopy 9 stress index, LF activity, HF activity, and HbA1c). Depression was measured based on subjective depression using a questionnaire, and sleep quality was measured based on average sleep hours and subjective sleep quality measured by the NRS. Immune response was measured based on IgG, CD4, CD8, and CD16+56 counts, which are physiological tests, and intersubject homogeneity was assessed at W0 (Tables 2 and 3). Subjective pre-treatment stress scores for the aromatherapy and waiting list groups were 6.48 points and 6.40 points, respectively. Pre-treatment physiological stress responses for the aromatherapy and waiting list groups, respectively, were as follows: a stress index of 4.53 13

and 4.23; LF activity of 5.35 and 5.55; HF activity of 5.06 and 5.36; HbA1c of 5.43% and 5.36%. There were no significant intergroup differences (Table 2). Pre-treatment subjective depression scores for the aromatherapy and waiting list groups were 34.77 points and 34.00 points, respectively. Mean sleep hours for the past week for the aromatherapy and waiting list groups were 6.05 hours and 6.33 hours, respectively, and subjective sleep quality for the past week was 5.48 points and 6.02 points, respectively. There were no significant differences between the groups (Table 2). In terms of physiological immune responses, IgG for the aromatherapy and waiting list groups were 1229.27 and 1249.60, respectively. Additionally, CD4 counts were 40.25 and 39.49, CD8 counts were 24.82 and 28.40, and CD16+56 counts were 12.64 and 10.56, respectively. There were no significant intergroup differences, with the exception of CD8 counts (Table 3). The pre-treatment testing of intergroup homogeneity for the dependent variables showed that only CD8 count (physiological immune response) was significantly different between the two groups. There were no significant intergroup differences in subjective stress scores, stress index, LF or HF activity, HbA1c, depression, mean sleep hours, subjective sleep quality, IgG, CD4, or CD16+56 (Tables 2 and 3). 3.2 The effects of aromatherapy on stress, sleep quality and immunity 3.2.1 Primary Outcomes Stress We measured subjective stress, stress index, LF activity, and HF activity three times: prior to treatment (W0), two weeks following treatment (W2), and four weeks following treatment (W4). HbA1c was measured two times: prior to treatment (W0) and four weeks 14

following treatment (W4). There were no significant differences between the aromatherapy and waiting list groups in pre-treatment (W0) subjective stress, stress index, LF or HF activity, or HbA1c (Table 2). A repeated measures ANOVA comparing subjective stress scores showed that there was a significant interaction between the two groups and time (F = 10.231, p < .001). Specifically, there was a significant difference in stress on the second week of treatment (W2), with 5.53 points and 6.57 points for the aromatherapy

and waiting list groups, respectively (t = -

2.332, p = .023). There was also a significant difference in stress on the fourth week of treatment (W4), with 3.53 points and 5.68 points for the aromatherapy

and waiting list

groups, respectively (t = -4.744, p < .001). For stress index, a repeated measures ANOVA showed there was no interaction between the two groups and time (F = 1.502, p = .227) as well as no significant differences between the two groups both on the second week of treatment (W2) and fourth week of treatment (W4). Similarly, a repeated measures ANOVA showed that there was no interaction between the two groups and time for LF and HF activity. There was no significant difference between the aromatherapy group (5.36%) and waiting list group (5.29%) in HbA1c after the fourth week of treatment (W4). Sleep Quality We measured average sleep hours and subjective sleep quality using the NRS three times. The results indicated that there was no significant difference in average hours of sleep or sleep quality between the aromatherapy and waiting list groups prior to treatment (W0).

15

A repeated measures ANOVA for average sleep length showed that there was a significant interaction between the two groups and time (F = 6.058, p = .003). In addition, post-hoc tests showed a significant difference on the second week of treatment (W2), with 6.02 hours and 5.93 hours of sleep for the aromatherapy and waiting list groups, respectively (F = 5.054, p = .028). There was also a significant difference in sleep after the fourth week of treatment (W4), with 6.33 hours and 6.07 hours of sleep for the aromatherapy

and waiting

list groups, respectively (F = 8.640, p = .005). A repeated measures ANOVA for sleep quality showed that there was a significant interaction between the two groups and time (F = 9.045, p < .001). In addition, post-hoc tests showed a significant difference in sleep quality on the second week of treatment (W2), with scores of 6.67 and 5.20 for the aromatherapy and waiting list groups, respectively (t = 3.194, p = .002). There was also a significant difference in sleep quality after the fourth week of treatment (W4), with scores of 7.10 and 5.22 for the aromatherapy and waiting list groups, respectively (t = 3.635, p = .001; Table 2).

Immunity We measured IgG, CD4, CD8, and CD 16+56 three times. Prior to treatment (W0), there were no significant differences between the aromatherapy

and waiting list groups in IgG,

CD4, and CD 16+56, but there was a significant difference in CD8 (t = -2.180, p = .033). CD8 for the aromatherapy and waiting list groups was 24.82 and 28.40, respectively. There were no significant differences between the groups in IgG, CD4, and CD8 after the second and fourth weeks of treatment. However, there was a significant difference in CD16+56 after the second week of treatment (W2), with values of 13.21 and 9.96 for the 16

aromatherapy

and waiting list groups, respectively (t = 2.215, p = .031). There was not a

significant difference between the groups after the fourth week of treatment (W4), with values of 14.55 and 13.47 for the aromatherapy and waiting list groups, respectively (t = 0.647, p = .520). Repeated measures ANOVAs for IgG, CD4, CD8, and CD 16+56 showed that there were no significant interactions between the two groups and time (Table 3).

3.2.2 Secondary outcome Depression To verify the effects of inhalation of aroma essential oils on depression, we measured depression using the CES-D in three sessions. There was no significant difference in depression prior to treatment (W0) between the aromatherapy (34.77 points) and control (34.00 points) groups (t = 0.391, p = .697). Repeated measures ANOVA showed a significant interaction between the two groups and time (F = 6.433, p = .002). Post-hoc tests showed that there was no significant difference on the second week of treatment (W2), with 31.83 points and 35.60 points for the aromatherapy and waiting list groups, respectively (t = -1.836, p = .071). However, there was a significant difference in depression on the fourth week of treatment (W4), with 29.77 points and 33.80 points for the aromatherapy and waiting list groups, respectively (t = -2.015, p = .049; Table 2). 4. Discussion This study sought to verify the effects of 4 weeks of inhalation aromatherapy on adults’ stress, depression, sleep quality, and immunity. Stress was divided into perceived stress and physiological stress (i.e., stress index, LF, HF, HbA1c), and immunity was measured based on immunologic indices (i.e., IgG, CD4, CD8, CD16+56). The experimental treatment 17

involved the inhalation of an essential oil blend of lemon, eucalyptus, tea tree, and peppermint in a ratio of 4:2:2:1 using a pendant and aroma stone for 4 weeks. The waiting list group did not receive any form of treatment until after data collection was complete in week 4. To assess the effects of inhalation aromatherapy, we examined changes in our measures from before treatment and after the second and fourth weeks of treatment. Stress refers to the psychological, physiological, and physical state of tension that is felt in a difficult situation [6]. In the present study, stress was divided into perceived and physiological. Perceived stress was measured using the NRS, while physiological stress was measured based on the stress index, ANS activation, and glycated hemoglobin using ANS measurement equipment. Subjective stress was significantly lower in the aromatherapy group than the waiting list group, but the two groups did not differ in terms of the stress index, ANS activation, or glycated hemoglobin. These results are comparable to the findings of previous studies, where aromatherapy inhalation of essential oil reduced perceived stress [29], and relieved stress and anxiety in pregnant women [30], high school students [31], and percutaneous coronary intervention (PCI) patients in the intensive care unit (ICU) [32]. Based on these findings, aromatherapy essential oil inhalation seems to be effective in relieving psychological stress and anxiety. However, there were no significant differences between the aromatherapy and waiting list groups in objective indicators of stress, including the stress index, ANS activation, and glycated hemoglobin levels. Previous studies have found aromatherapy to be both effective [33] and ineffective [34], rendering it difficult to draw concrete conclusions on its effectiveness. Additionally, glycated hemoglobin levels reflect blood sugar levels within the past 3 months, but there was a 2-month overlap in the present study, further clouding the results. Therefore, while inhalation aromatherapy is believed to 18

relieve psychological stress via various psychological effects, this study could not verify this theory using objective indices. In addition to reducing perceived stress, inhalation aromatherapy using essential oils was also effective in mitigating depression after the fourth week of treatment. This result fits with the findings of a meta-analysis of the psychosocial effects of aromatherapy from a nursing intervention [35] that reported massage and inhalation aromatherapy were effective in reducing depression. Moreover, this present study also measured sleep length and quality (NRS) to examine the effects of aroma essential oil inhalation on sleep. There were no significant differences between the two groups prior to treatment on sleep length or sleep quality (NRS). However, after the second and fourth weeks of aromatherapy treatment, the aromatherapy group had significantly longer sleep and better quality sleep than the waiting list group. These results are similar to those of a study that reported aroma oil inhalation increased the quality of sleep of patients who received percutaneous coronary intervention (PCI) intervention [32]. Similarly, another investigation reported that aroma inhalation increased objective sleep patterns and subjective sleep quality, excluding sleep length, in night shift nurses [36]. Furthermore, the present study also sought to examine the effects of aromatherapy using essential oil inhalation on immunity by measuring IgG, CD4, CD8, and CD16+56 counts. IgG accounts for the vast majority (80%) of all immunoglobulins and plays an important role in helping natural killer (NK) cells to detect and destroy targets, such as microorganisms or tumor cells. CD4 secretes cytokines in response to external infection and helps activate CD8, which in turn displays cytotoxicity towards infected host cells. CD16+56 is expressed by NK cells, and NK cells are involved in the early immune response [37]. Our results showed that 19

there were no significant differences in immunity between the groups prior to treatment, or after the second and fourth weeks of aromatherapy treatment. While our study did not find differences in immunity after aroma essential oil inhalation, there have been several studies that found immune-enhancing effects of aromatherapy. In one study that examined college students, the group of students who received aromatherapy massage using lavender, cypress, and sweet marjoram oil had statistically significant increases in CD8 and CD16 compared to their levels prior to aromatherapy [38]. In the same study, applying aromatherapy with light Thai massage in colorectal cancer patients undergoing chemotherapy resulted in significant increases in lymphocyte count, although there were no statistically significant changes in CD4, CD8, and CD4/CD8 [38]. Moreover, in another study that examined the effects of aromatherapy in college students with tension headaches, there were no significant differences in the T cell and NK cell ratios between the aromatherapy and waiting list groups. As shown here, prior studies have reported contrasting results with regard to the immuneenhancing effects of aromatherapy, which suggests a need for more studies in the future. The limitations of this study are as follows. A limitation of this study is that it was not previously registered in a clinical trial registry, however, the study was conducted according to the strict criteria described. There was no placebo control group and the study was unblinded. In addition, the value of HbA1c of the subjects were in the normal range and therefore to identify a change as a result of the aromatherapy intervention would have been unlikely. In particular, the immunoassay was unable to carry out all of the immunoassay tests, and only a few tests were performed. Although the sample size was powered to detect a significant difference, it is difficult to make be conclusive with one experiment with 30 subjects. Therefore, we hope to repeat a rigorous experimental study in the future. 20

In this study, aroma oil inhalation has no effect on the immune system in adults. However, since it has the effect of alleviating stress and depression and improving the quality of sleep, it is a suitable intervention method to manage stress and depression and improve the quality of sleep I think. If you inhale the aroma oil blended by the experts as usual in this study protocol, stress and depression that are common to adults will be alleviated and sleep disorders will be improved.

5. Conclusions In conclusion, aromatherapy via essential oil inhalation was effective in mitigating perceived stress and depression, and improved sleep quality. The results of this experiment using essential oil inhalation showed that the subjects were effective in reducing the perceived stress, depression, and in increasing the sleep quality but not in the objective stress index reduction and immune enhancement effect. However, its immunity-enhancing effects were not verified and require further rigorous investigation. In the next study, we should establish a placebo control group and a control group, and set the experimental study period to reflect the blood glucose level of HbA1C, one of the physiological stress items. The number of subjects should be increased.

Conflict of Interest none Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1A1A3A04001441)

21

References [1].

National Cancer Information Center. Incidence od cancer [internet] 2017 [cited 2017 April14]

[Available from: http://www.cancer.go.kr/mbs/cancer/subview.jsp?id=cancer_040101000000. [2].

Centers for Disease Control and Prevention. Disease information 2017 [cited 2017 April14]

[Available from: http://cdc.go.kr/CDC/contents/sub1.jsp. [3].

Gomez-Merino D, Drogou C, Chennaoui M, Tiollier E, Mathieu J, Guezennec CY. Effects of

combined stress during intense training on cellular immunity, hormones and respiratory infections. Neuroimmunomodulation. 2005;12(3):164-72. [4].

Jin S, Whang YM, Chang IH. Genetically Modified Bacteria as Targeted Agent for Cancer.

The Korean Journal of Urological Oncology. 2016;14(2):54-62. [5].

Rodgers S, Maloney B, Ploderer B, Brereton M, editors. Managing stress, sleep and

technologies: an exploratory study of Australian university students. Proceedings of the 28th Australian Conference on Computer-Human Interaction; 2016: ACM. [6].

Schneiderman N, Ironson G, Siegel SD. Stress and health: psychological, behavioral, and

biological determinants. Annual review of clinical psychology. 2005;1:607. [7].

Wikipedia. Depression(mood) [Internet]. 2016 [cited 2016 July 6] [Available from: https://en.

wikipedia.org/wiki/Depression_(mood). [8].

Kryger M, Roth T, Dement W. Principles and practice of sleep medicine. 4th ed. Philadelphia:

Elsevier Health Sciences; 2005. [9].

The Korean Society of Biosociobehavioral Science. Scientific understanding of stress. Seoul:

shinkwangpub; 1997. [10].

wikipedia. Sleep [Internet]. 2015 [cited 2016 January 12] [Available from: https://en.wikipe

dia.org/wiki/Sleep. [11].

Sukegawa T, Itoga M, Seno H, Miura S, Inagaki T, Saito W, et al. Sleep disturbances and

depression in the elderly in Japan. Psychiatry and Clinical Neurosciences. 2003;57(3):265-70. [12].

Ancoli‐Israel S, Cooke JR. Prevalence and comorbidity of insomnia and effect on functioning

in elderly populations. Journal of the American Geriatrics Society. 2005;53(S7):S264-S71. [13].

Kim YG, Kim TW, Kim MH, Kim AN, Kim HY, Kim HJ, et al. Medical microbiologys &

infectious disease. Seoul: soomoonsa; 2011. [14].

Curry JM, Hanke ML, Piper MG, Bailey MT, Bringardner BD, Sheridan JF, et al. Social

disruption induces lung inflammation. Brain, behavior, and immunity. 2010;24(3):394-402. [15].

Irwin MR, Levin MJ, Carrillo C, Olmstead R, Lucko A, Lang N, et al. Major depressive

disorder and immunity to varicella-zoster virus in the elderly. Brain, behavior, and immunity. 22

2011;25(4):759-66. [16].

Miller GE, Cohen S, Pressman S, Barkin A, Rabin BS, Treanor JJ. Psychological stress and

antibody response to influenza vaccination: when is the critical period for stress, and how does it get inside the body? Psychosomatic medicine. 2004;66(2):215-23. [17].

Lawless J. The complete illustrated guide to aromatherapy: a practical approach to the use of

essential oils for health and well-being: HarperCollins UK; 1997. [18].

Hur M-H, Song J-A, Lee J, Lee MS. Aromatherapy for stress reduction in healthy adults: a

systematic review and meta-analysis of randomized clinical trials. Maturitas. 2014;79(4):362-9. [19].

Field T, Field T, Cullen C, Largie S, Diego M, Schanberg S, et al. Lavender bath oil reduces

stress and crying and enhances sleep in very young infants. Early human development. 2008;84(6):399-401. [20].

Nord D, Belew J. Effectiveness of the essential oils lavender and ginger in promoting

children's comfort in a perianesthesia setting. Journal of PeriAnesthesia Nursing. 2009;24(5):307-12. [21].

Saiyudthong S, Marsden CA. Acute effects of bergamot oil on anxiety‐related behaviour and

corticosterone level in rats. Phytotherapy Research. 2011;25(6):858-62. [22].

Baker J, Brown K, Rajendiran E, Yip A, DeCoffe D, Dai C, et al. Medicinal lavender

modulates the enteric microbiota to protect against citrobacter rodentium-induced colitis. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2012;303(7):G825-G36. [23].

Khiewkhern S, Promthet S, Sukprasert A, Eunhpinitpong W, Bradshaw P. Effectiveness of

aromatherapy with light thai massage for cellular immunity improvement in colorectal cancer patients receiving chemotherapy. Asian Pacific Journal of Cancer Prevention. 2013;14(6):3903-7. [24].

McDowell I. Measuring health: a guide to rating scales and questionnaires: Oxford university

press; 2006. [25].

Svensson E. Comparison of the quality of assessments using continuous and discrete ordinal

rating scales. Biometrical Journal. 2000;42(4):417-34. [26].

Jun JS, Kwon JH, Kim MS. Durative effects of the stress management group psychotherapy

on glycemic control in type 2 diabetes: SMBG Profile & PMR. The Korean Journal of Health Psychology. 2010;15(1):51-66. [27].

Cho MJ, Kim KH. The diagnostic validity of the CES-D(Korean version) in the assessment

of DSM-III-R major depression J Korean Neuropsychiatr Asspc. 1993;32(3):381-99. [28].

Ali B, Al-Wabel NA, Shams S, Ahamad A, Khan SA, Anwar F. Essential oils used in

aromatherapy: A systemic review. Asian Pacific Journal of Tropical Biomedicine. 2015;5(8):601-11. [29].

Toda M, Morimoto K. Effect of lavender aroma on salivary endocrinological stress markers.

Archives of oral biology. 2008;53(10):964-8. 23

[30].

Igarashi T. Physical and psychologic effects of aromatherapy inhalation on pregnant women:

a randomized controlled trial. The Journal of Alternative and Complementary Medicine. 2013;19(10):805-10. [31].

Seo J-Y. The effects of aromatherapy on stress and stress responses in adolescents. Journal of

Korean Academy of Nursing. 2009;39(3):357-65. [32].

Cho M-Y, Min ES, Hur M-H, Lee MS. Effects of aromatherapy on the anxiety, vital signs,

and sleep quality of percutaneous coronary intervention patients in intensive care units. EvidenceBased Complementary and Alternative Medicine. 2013;2013. [33].

Chang K-M, Shen C-W. Aromatherapy benefits autonomic nervous system regulation for

elementary school faculty in Taiwan. Evidence-Based Complementary and Alternative Medicine. 2011;2011. [34].

Chien L-W, Cheng SL, Liu CF. The effect of lavender aromatherapy on autonomic nervous

system in midlife women with insomnia. Evidence-Based Complementary and Alternative Medicine. 2011;2012. [35].

Roh K, Park H-A. A Meta-Analysis of the Effects of Aromatherapy on Psychological

Variables in Nursing. Journal of Korean Academy of Community Health Nursing. 2009;20(2):113-22. [36].

Chnag Soon B, Chu Sang H, Kim Yeong I, Yun Sun H. The effects of aroma inhalation on

sleep and fatigue in night shift nurses. Korean Journal of Adult Nursing. 2008;20(6):941-9. [37].

Coico R, Sunshine G, Benjamini E. Immunology. Hoboken, N.J.: John Wiley; 2008.

[38].

Kuriyama H, Watanabe S, Nakaya T, Shigemori I, Kita M, Yoshida N, et al. Immunological

and psychological benefits of aromatherapy massage. Evidence-Based Complementary and Alternative Medicine. 2005;2(2):179-84.

24

Fig.1

Fig.2

25

Table 1. Intergroup homogeneity in baseline characteristics of subjects Characteristics/

Aromatherapy

Waiting list control

Variables

Group

Group

(n = 30)

(n = 30)

t /χ²

p

Mean ± SD or N (%) Mean ± SD or N (%) Age (years)

37.30 ± 6.75

35.83 ± 6.88

0.833

.408

Height (cm)

159.63 ± 3.72

159.23 ± 3.93

0.405

.687

Body weight (kg)

56.20 ± 9.07

54.37 ± 6.65

0.893

.376

Married

20 (66.7)

18 (60.0)

0.287

.592

Single

10 (33.3)

12 (40.0)

College

24 (80.0)

25 (83.3)

0.111

.739

Graduate school

6 (20.0)

5 (16.7)

Day fixed work

26 (86.7)

25 (83.3)

0.131

.718

4 (13.3)

5 (16.7) 1.818

.403

0.351

.554

0.480

.488

2.013

.366

6.312

.053*

4.286

.112*

Marital status

Education

Working form

Shift work Monthly income

<300

11 (36.7)

9 (30.0)

(10,000 KRW/M)

300–599

11 (36.7)

16 (53.3)

8 (26.6)

5 (16.7)

Yes

29 (96.7)

28 (93.3)

No

1 (3.3)

2 (6.7)

No

26 (86.7)

24 (80.0)

Yes

4 (13.3)

6 (20.0)

No

5 (16.7)

5 (16.7)

≥600 Caffeine

Disease

Drinking alcohol

Exercise

Immune disease

Sleep disturbance

Less than 1/month 9 (30.0)

14 (46.7)

More than 1/month16 (53.3)

11 (36.6)

No

12 (40.0)

21 (70.0)

< 2 times/week

13 (43.3)

8 (26.7)

> 3 times/week

5 (16.7)

No

30 (100.0)

Yes

0 (0.0)

No

26 (86.7)

Yes

4 (13.3)

1 (3.3)

0 (0.0) 30 (100.0) 0 (0.0)

Mean ± SD = Mean ± Standard Deviation *

30 (100.0)

Fisher’s exact test

26

Table 2. The effects of aromatherapy on stress, depression, and sleep between aromatherapy group and waiting list control group. Variable

Stress

Aromatherapy Group (n = 30) Mean ± SD

Waiting list control Group (n = 30) Mean ± SD

Perceived W0 stress score W2

6.48 ± 1.96

6.40 ± 1.43

0.188

.851

5.53 ± 1.87

6.57 ± 1.55

-2.332

.023

W4

3.53 ± 1.98

5.68 ± 1.50

-4.744

<.001

Stress W0 index W2 (canopy 9) W4

4.53 ± 2.56

4.23 ± 2.37

0.471

.639

4.97 ± 2.53

4.83 ± 2.28

0.215

.831

3.97 ± 2.03

4.77 ± 2.05

-1.522

.133

LF activity W0

5.35 ± 1.16

5.55 ± 1.14

-0.680

.499

W2

5.30 ± 1.04

5.39 ± 1.18

-0.342

.733

W4

5.58 ± 0.86

5.17 ± 0.92

1.799

.077

W0 W2 W4 W0

5.06 ± 1.50 4.67 ± 1.35 5.21 ± 1.06 5.43 ± 0.45

5.36 ± 1.26 5.12 ± 1.18 5.16 ± 0.98 5.36 ± 0.25

-0.827 -1.383 0.200 0.777

.412 .172 .842 .441

W4

5.36 ± 0.40

5.29 ± 0.27

0.827

.411

W0 W2

34.77 ± 7.94 31.83 ± 7.60

34.00 ± 7.22 35.60 ± 8.27

0.391 -1.836

.697 .071

W4

29.77 ± 8.05

33.80 ± 7.44

-2.015

.049

W0

6.05 ± 1.06

6.33 ± 0.90

-1.130

.263

W2

6.02 ± 0.92

5.93 ± 0.92

5.054

.028

W4

6.33 ± 1.04

6.07 ± 0.93

8.640

.005

Quality of W0 sleep W2 (QOS) W4

5.48 ± 2.06

6.02 ± 1.67

-1.100

.276

6.67 ± 1.84

5.20 ± 1.71

3.194

.002

7.10 ± 2.12

5.22 ± 1.88

3.635

.001

HF activity HbA1c

Depression

Sleep

Time of sleep (hours/ day)

nd

th

t / F* p

W0 = Pretest day; W2 = Experimental 2 week; W4 = Experimental 4 week; G = group; T = time Mean ± SD= Mean ± Standard Deviation *ANCOVA † Repeated measures ANOVA

27

F (p)†

Time: 30.593 (< .001) G*T: 10.231 (< .001) Group: 9.051 (.004) Time: 1.572 (.212) G*T: 1.502 (.227) Group: 0.075 (.785) Time: 0.187 (.830) G*T: 1.880 (.157) Group: 0.041 (.840) Time: 2.006 (.145) G*T: 1.054 (.346) Group: 0.907 (.345) Time: 19.496 (< .001) G*T: 0.000 (> .999) Group: 0.659 (.420) Time: 6.449 (.002) G*T: 6.433 (.002) Group: 1.684 (.199) Time: 4.929 (.009) G*T: 6.058 (.003) Group: 0.008 (.927) Time: 0.907 (.407) G*T: 9.045 (< .001) Group: 7.659 (.008)

Table 3. The effects of aromatherapy on immunity between aromatherapy group and waiting list control group. Variable

Immune

IgG

CD4

CD8

CD

Aromatherapy

Waiting list control

Group (n = 30)

Group (n = 30)

Mean ± SD

Mean ± SD

t / F*

p

F (p) †

W0 1229.27 ± 141.28

1249.60 ± 179.49

-0.488

.628

Time: 57.454 (< .001)

W2 1263.33 ± 169.12

1281.50 ± 183.86

-0.398

.692

G*T: 0.143 (.828)

W4 1325.80 ± 204.23

1353.70 ± 205.59

-0.527

.600

W0 40.25 ± 6.58

39.49 ± 7.06

0.430

.669

Time: 12.128 (< .001)

W2 40.99 ± 6.98

40.88 ± 7.68

0.056

.955

G*T: 0.337 (.715)

W4 36.88 ± 6.03

37.43 ± 6.44

-0.337

.737

W0 24.82 ± 5.72

28.40 ± 6.94

-2.180

.033

Time: 1.564 (.218)

W2 24.49 ± 6.10

27.57 ± 6.48

0.100

.753

G*T: 1.664 (.202)

W4 26.30 ± 6.09

27.83 ± 7.01

0.491

.486

W0 12.64 ± 6.43

10.56 ± 6.03

1.291

.202

Time: 7.850 (.002)

9.96 ± 4.61

2.215

.031

G*T: 1.191 (.300)

13.47 ± 5.69

0.647

.520

16+56 W2 13.21 ± 6.59 W4 14.55 ± 7.16

Group: 0.235 (.630)

Group: 0.005 (.944)

Group: 3.277 (.075)

Group: 2.466 (.122)

W0 = Pretest day; W2 = Experimental 2nd week; W4 = Experimental 4th week; G = group; T = time; IgG = immunoglobulin; CD = cluster of differentiation Mean ± SD= Mean ± Standard Deviation *ANCOVA †Repeated

measures ANOVA

28