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Research Article
The Effect of Copaiba Oil Odor on Anxiety Relief in Adults under
Mental Workload: A Randomized Controlled Trial
Nan Zhang ,
1
,
2
Jie Chen ,
1
,
2
Wenyan Dong ,
2
and Lei Yao
1
,
3
1
School of Design, Shanghai Jiao Tong University, 800 Dong Chuan Rd., Shanghai, China
2
Aromatic Plant R&D Center, Shanghai Jiao Tong University, 800 Dong Chuan Rd., Shanghai, China
3
d�oTERRA (Shanghai) Commercial Co., Ltd, 699 West Nanjing Rd., Shanghai, China
Correspondence should be addressed to Lei Yao; yaolei@sjtu.edu.cn
Received 10 November 2021; Revised 9 February 2022; Accepted 21 February 2022; Published 20 March 2022
Academic Editor: Teh Lay Kek
Copyright ©2022 Nan Zhang et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Aromatherapy has been proved to be effective in alleviating anxiety in practices and research. Recently, copaiba oil
(CPO) is popular in the market and is recommended for anxiety relief in aromatherapy practice. However, relevant scientific
research is still lacking. Methods. A randomized controlled trial was designed to evaluate the anxiety-relieving effect of CPO
inhalation in 22 adults. Jojoba oil was used as the control treatment. N-back and mental arithmetic tasks were used as stress
stimulation. CPO or control intervention was carried out after the n-back training phase. e State-Trait Anxiety Inventory
(STAI), EEG activities, physiological indexes including heart rate (HR), blood pressure (BP), blood oxygen saturation, and salivary
cortisol were assessed in different phases of the experimental process. Results. ere was no significant difference in the change of
HR and BP between the CPO and control groups before odor intervention. e S-AI scores of the CPO treated participants
decreased after the n-back and mental arithmetic tests and were significantly lower than those of the participants who received
control treatments. e HR and salivary cortisol of participants who received CPO intervention significantly decreased during the
n-back and mental arithmetic tests. Furthermore, a remarkable decrease of beta wave activity was observed in the left midfrontal
region (F3) when the participant received the CPO intervention. Conclusion. e study’s findings supported that the CPO odor
showed beneficial effects on alleviating anxiety based on several indicators in subjective, physiological, and EEG measurements.
1. Introduction
Essential oils are derived from aromatic plants and are
composed of various volatile chemical components. In
aromatherapy, odor molecules enter the nasal cavity and
attach to specific olfactory receptors on olfactory neurons.
Such combination generates action potentials, initiates
signal transduction, and further affects the neural activities
in the limbic system that are closely related to anxiety and
depression regulation [1]. In addition, essential oil com-
pounds can easily enter the blood circulation by inhalation
or massage application because of their small molecular
weights and strong permeability.
In aromatherapy practices, the use of some essential oils
could quickly relieve anxiety, stress, or other negative
emotions [2]. At present, many studies provided evidence
for the anti-anxiety effects of essential oils from citrus fruit
peels [3], lavender [4], rose [5], and so on through either
animal tests or clinical trials. In the history of the aroma-
therapy practice, resinous essential oils are also considered
to be beneficial to the nervous system. However, there is little
research on the emotion regulation effect of resin essential
oils. Frankincense extracts have been proved to be anxiolytic
in a previous preclinical test [6].
Copaiba balsam is the resin of the Copaidera genus,
which is common in the Amazon region. Copaiba oil (CPO)
is extracted from its resin by hydrodistillation. e content
of β-caryophyllene could reach more than 50% in the CPO
[7]. Because of its anti-inflammatory [8], skin-healing-
promoting effect [9], and anti-parasitic properties [10], CPO
has been widely used in folk medicine. e relevant phar-
macological properties have been confirmed by modern
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2022, Article ID 3874745, 9 pages
https://doi.org/10.1155/2022/3874745
scientific research in recent years. For example, CPO (3%)
cream application could effectively prevent mechanical
tenderness induced by UVB radiation in mice [7]. When
given the CPO to volunteers suffering from hand arthritis by
massage, the time of subjects completing dexterity tasks was
significantly reduced, and the pain score decreased by ap-
proximately 50% compared to the control treatment [11].
β-Caryophyllene, which is the main component of CPO,
has been proved to be anxiolytic in many previous studies
[12]. It was thought to be a CB2 receptor, potent-selective
agonist. erefore, it could be speculated that CPO might
have the function of anxiety-relieving. A recent animal study
showed that intraperitoneal administration of CPO had an
acute anxiolytic effect [13]. However, there is no evidence
suggesting that the inhalation of the CPO has an anti-anxiety
effect.
Cortisol produced in the adrenal cortex is the main
glucocorticoid in the human body. It is released by the
hypothalamic-pituitary-adrenal axis in response to various
psychological stimuli. Many research had proved that es-
sential oils with anxiolytic effects could attenuate the plasma
corticosterone in animals [14] and decrease the salivary
cortisol in clinical trials [15, 16]. Electroencephalogram
(EEG) has a more direct relationship with brain activity. It
has been used to study the relationship between olfactory
stimulation and emotion. Several EEG studies have dem-
onstrated significant alterations in activities of alpha waves
(7.5–12.5Hz) or beta waves (12.5–30Hz) after the inter-
vention of essential oils [17, 18]. In addition, heart rate (HR)
and blood pressure (BP) are often considered to be related to
anxiety. In many clinical studies of essential oils, these two
indicators were positively correlated with anxiety [19, 20].
erefore, the aim of this study was to investigate the
efficacy of CPO inhalation for anxiety-relieving in a clinical
trial. N-back and mental arithmetic tasks were carried out as
mental workload and stress stimulation. e State-Trait
Anxiety Inventory (STAI) was used as a self-report of
anxiety. EEG, salivary cortisol, as well as several physio-
logical indexes were recorded during the experiment.
2. Materials and Methods
2.1. Participants and Randomization. A randomized con-
trolled trial was designed to evaluate the anxiety-relieving
effect of inhalation of CPO. Jojoba oil, an almost odorless oil,
was used as the control treatment. e study was approved
by the Research and Ethics Offices of the Shanghai Jiao Tong
University (No. H2021148I).
e study was conducted on the campus of Shanghai Jiao
Tong University. Subjects were recruited through social-
oriented electronic questionnaires. Subjects were eligible if
they were aged 18–40 years old, with no nasal diseases, no
medical conditions that could affect the sense of smell, no
history of allergy to essential oils, and no history of taking
psychotropic drugs in recent three months. Furthermore,
female subjects should not be in the stage of preparation for
pregnancy or pregnancy to avoid any potential affection by
the odors. e informed consent forms were signed by the 22
eligible participants. e subjects (5 men and 17 women)
were randomly assigned to the CPO group (n�11) and the
control group (n�11). Practitioner and participant blinding
were not considered possible, due to the nature of the odor
intervention. e participants were not informed of when
the odor was released in the experiment.
2.2. Materials and Intervention. CPO was provided by the
doTERRA (Shanghai) Commercial Co. Ltd. Jojoba oil was
provided by the Aromatic Plant R&D Center of Shanghai
Jiao Tong University. An incense diffuser that could directly
atomize pure essential oil and control the release time was
used.
A separate space (1.7 ×1.7 ×1.85 m) was used as the test
space. A chair and a small table were placed in this test space.
e subject could finish the questions on a computer screen
placed on the table. e incense diffuser was placed on the
ground of the corner so that subjects would not notice it
easily. When entering the aromatherapy intervention
progress, the experimenter will start the incense diffuser and
atomize the copaiba oil into the air. e working mode of the
incense diffuser was set at every 60 s followed by a pause for
5 sec.
2.3. N-Back and Mental Arithmetic Tasks. e n-back task.
N-back test is a kind of mental load task [21], in which
subjects are asked to monitor a series of briefly presented
stimuli and decide in each trial if the currently presented
stimuli were the same as those presented one or more trials
before [22]. In this study, a 300-s 1-back task was used as
stress stimulation. All participants performed the 1-back
task two times, for the first time as a training session, and the
second time as a test session. e n-back task field was a
square separated into smaller squares with lines (3 ×3
squares) shown on the computer screen. During the task, a
visual stimulus accompanied with an auditory stimulus
(a single letter) was randomly presented 500 ms in one of the
squares every 3 s. e subjects should find a match of the
position and color of the current image and the letter sound
in the auditory to those shown one step earlier. e task was
held through an online website (https://brainscale.net/dual-
n-back). e participants were prompted on the computer
screen if they choose a wrong answer.
2.3.1. Mental Arithmetic Task. e participants were re-
quired to finish a 5 min mental arithmetic task containing 30
trials of 3-digit addition on the computer screen. Each trial
lasts 10 s. e task was held through an online website
(https://brainscale.net/mental-math). e participants
would be prompted on the computer screen if they filled in a
wrong answer.
2.4. Measurements of Physiological Index and Saliva Cortisol.
HR, systolic blood pressure (SBP), and diastolic blood
pressure (DBP) were measured by a digital blood pressure
monitor (Omron). Blood oxygen saturation (SpO
2
) was
measured by a finger clip oxygen saturation tester (Heal
Force).
2Evidence-Based Complementary and Alternative Medicine
e saliva of the subjects was collected by EP tube and
centrifuged at 8,000 rpm, 4°C for 10 min. e supernatant
was collected and stored at −80°C. A human cortisol Elisa kit
(Elabscience, China) was used to evaluate the cortisol
content in the saliva. All operations were carried out strictly
according to the instructions of the kit.
2.5. EEG Recording and Spectrum Analysis. e EEG was
recorded using the eego™mylab system (eego miniEE-401,
ANT Neuro, Inc.). A cap (waveguard) was positioned on the
subject using the standard 10–20 system. e electrode gel was
applied onto electrode sites that were located at midfrontal (F3
and F4), central (C3 and C4), Fpz, Cz, and Pz. FCz was used as
the reference electrode. Impedances were brought below
10K ohms. EEG signals were acquired at a sampling rate of
500 Hz. EEG data were digitally filtered offline to a 1–30 Hz
bandwidth. e EEG data were edited for artifact using soft-
ware asalab 4.10.2. Separate EEG relative power of alpha and
beta spectral bands in the midfrontal and central brain regions
were analyzed. Alpha and beta wave activities were highly
related to anxiety mood change [17, 23]. Previous studies re-
ported that brain activity in the prefrontal cortex was strongly
linked to anxiety [24], and excessive power at beta frequencies
around electrode sites C3, CZ, and/or C4 could be observed
when both anxiety and problems with attention occurred [25].
erefore, the relative power of each of the alpha (7.5–12.5 Hz)
and beta (12.5–30 Hz) spectral bands was expressed as a
percentage (%) of the total spectral power within the 1–30 Hz
window, as previously reported [26].
2.6. Self-Report Measures. e STAI was compiled by
Charles D. Spielberger et al. e first edition was published
in 1970 and translated into Chinese in 1988. e scale is a
self-assessment scale, which consists of 40 descriptive
questions and is divided into 2 subscales: (1) state anxiety
scale (S-AI), including questions 1–20, which describes a
usually transient unpleasant emotional experience, such as
tension, fear, anxiety, and neuroticism, accompanied by
hyperfunction of the nervous system and (2) trait anxiety
scale (T-AI), including questions 21–40. Trait anxiety de-
scribes a relatively stable anxiety tendency as a personality
trait with individual differences [27]. In this study, the STAI
was used to measure the anxiety state of the participant.
A Likert scale ranging from −5 to 5 was used to self-
evaluate the comfort of the environment odor in the test
zoom at the end of the experimental procedure. A score of
“5” to “−5” was assigned to the feeling of “very comfortable”
to “very uncomfortable.” A five-level scale was used to self-
evaluate the intensity of the environment odor. Scores of
1–5, respectively, represent “no smell,” “almost unrecog-
nizable smell,” “slightly identifiable smell,” “easily identifi-
able smell,” and “strong smell.”
2.7. Experimental Procedure. e subjects were asked to avoid
alcohol, nicotine, coffee, drugs, and any essential oil on the test
day. Food intake was not allowed 1.5 h before the test. At the
beginning of the test, subjects were asked to sit in a comfortable
position. e EEG cap was positioned on the subject’s head.
After an adaptive sitting for 10 min, the subjects completed the
baseline physiological parameters measurements including
HR, BP, and blood oxygen saturation. Saliva was also collected.
e EEG of the subject was recorded for 5 min with eyes closed.
e experimental procedure (Figure 1) was conducted in
the following order. (1) e subjects were first given a 5min
training session of the n-back (n�1) task. After training, the
relevant physiological parameters were measured; saliva was
collected; and the STAI scale was filled out. (2) en, the EEG
was recorded for 10 min with the subject’s eyes closed. After
5 min, the odor of the essential oil or the jojoba oil was released
into the space through an incense diffuser without informing
the subject. e odor was released for a total of 20 min with a 5 s
stop between every 60 sec. (3) Subjects then performed a 5 min
n-back (n�1) test and a 5 min rapid mental arithmetic task.
e test results were given to the subjects after each task. en,
the EEG of the subject was recorded for 5 min with eyes closed.
e relevant physiological parameters of subjects were mea-
sured again. Saliva was also collected. e STAI scale and
environmental odor evaluation were filled out. All the work
tasks and STAI scales were performed on a computer.
2.8. Identification of Constituents of the Copaiba Oil. e
copaiba oil was diluted 10 times with a solution of ethanol and
n-hexane (1:1, v/v). Gas chromatography/mass spectrometer
(GC/MS, Agilent 7890B-5977 A) was used to analyze the
constituents of copaiba oil. e GC was filled with a meth-
ylpolysiloxane nonpolar column (DB-WAX: 30 m ×0.25 mm
×0.25 μm). e GC conditions were as follows: carrier gas,
helium (1 mL/min); split rate, 10:1; and column temperature,
50°C for 3 min, 50°C to 120°C at 4°C/min, then 120°C for
10 min, 120°C to 220°C at 2°C/min, then 220°C for 12 min. e
MS conditions were as follows: inlet line temperature, 280°C;
source temperature, 230°C; and mass spectra electron impact,
70 eV. Individual components were identified from the mass
spectral library (NIST14).
2.9. Statistical Analysis. Statistical analyses were conducted
using SPSS 14.0 software. Unpaired two-tailed Student’s
t-test was used when comparing data of subjects in different
groups. Paired two-tailed Student’s t-test was used when
comparing data collected at different times of the same
subject. Statistical significance was defined if pvalue <0.05.
3. Results
3.1. Chemical Composition of the Copaiba Oil. Terpenes were
the main ingredients of the CPO (Table 1). e CPO con-
tained 55.93% caryophyllene, 10.41% α-copaene, 6.81%
trans-α-bergamotene, and 5.46% humulene. e sum of the
relative contents of the compounds (relative content above
1%) was 93.03%.
3.2. e STAI Score Change after the CPO Intervention.
e results of STAI showed that the CPO odor environment
could well alleviate the anxiety of participants compared to
Evidence-Based Complementary and Alternative Medicine 3
the control odor environment under the same stress task
stimulation (Table 2). e STAI, S-AI, and T-AI scores of the
two groups showed no statistically significant difference after
the n-back training (p>0.05). Overall, compared with the
n-back training session, the tension of participants in each
group in the n-back and mental arithmetic test session
showed a decreased trend. e S-AI score of the CPO group
was significantly lower than the control group (p<0.05).
e STAI, S-AI, and T-AI scores of the CPO group sig-
nificantly decreased after the mental arithmetic test
(p<0.05). Only the T-AI score decreased significantly after
the mental arithmetic test in the control group (p<0.05).
Pre- and posttests mean the time point after n-back
training and after the mental arithmetic test, respectively.
e value represents means ±SEM. Paired two-tailed Stu-
dent’s t-test was used for the comparison between pre- and
posttest. Unpaired two-tailed Student’s t-test was used for
the comparison between the control and CPO group.
∗p<0.05 compared with the score of the participant before
the task test. #p<0.05 compared with the score of the
control group in the same experiment step.
3.3. e Physiological Change after the CPO Intervention.
CPO odor had no statistically significant effect on partici-
pants’ BP (Table 3). However, it could significantly affect the
HR of the participant. During the n-back and mental
arithmetic test sessions, the HR of the CPO group decreased,
and the change was significant compared to the control
group (p<0.05). SpO
2
levels of the CPO group showed an
upward trend during the n-back and mental arithmetic
sessions (Table 3).
e salivary cortisol level of the CPO group significantly
decreased from 374.0 ng/ml to 130.4 ng/ml (p<0.05) during
the n-back and mental arithmetic test sessions. On the
contrary, salivary cortisol of the control group showed an
upward trend (Figure 2). e salivary cortisol level of the
Adapt to the
environment
10 min
EEG
recording
(pre training)
EEG
recording
(post training)
EEG
recording
(intervention)
EEG
recording
(post task)
Training session Test session
5 min 5 min 5 min 5 min 5 min 5 min 5 min
N-back N-back Mental
arithmetic
Odor intervention
Physiological measurements
Saliva collection (s1)
Pre-test physiological measurements
Saliva collection (s2)
STAI self-report
Post-test physiological measurements
Saliva collection (s3)
STAI self-report
Evaluation of environmental odor
Figure 1: e experimental procedure.
Table 1: Main chemical compounds of the CPO and its released
odor in the air.
Retention time Compound Relative content (%)
20.07 Caryophyllene 55.93
16.83 α-Copaene 10.41
19.83 trans-α-Bergamotene 6.81
22.27 Humulene 5.46
23.81 Germacrene D 4.38
26.06 δ-Cadinene 2.01
24.82 β-Bisabolene 1.98
15.78 α-Cubebene 1.84
21.34 c-Elemene 1.55
16.15 c-Muurolene 1.34
23.07 δ-Elemene 1.32
Sum 93.03
e table only lists the compounds with a relative content above 1%.
Table 2: e STAI score change after CPO or control treatment.
Control group (n�11) CPO group (n�11)
Pretest Posttest Pretest Posttest
STAI score 87.1 ±5.8 82.8 ±5.9 78.7 ±4.0 72.9 ±3.6∗
S-AI score 37.9 ±3.2 36.5 ±3.7 33.5 ±1.8 32.3 ±1.6∗#
T-AI score 49.2 ±3.2 46.3 ±3.1∗45.2 ±3.0 40.6 ±3.1∗
Table 3: Comparison of the change of four physiological indexes
between control and CPO group in n-back training and n-back test
sessions.
Training
session Test session
SBP change (%)
Control group
(n�11) 1.23 ±1.64 −0.71 ±1.21
CPO group (n�11) 4.55 ±1.28 −1.31 ±1.83
pvalue 0.13 0.79
DBP change
(%)
Control group
(n�11) 3.51 ±2.84 −0.60 ±2.23
CPO group (n�11) 4.06 ±2.46 −1.12 ±1.52
pvalue 0.88 0.85
HR change (%)
Control group
(n�11) −3.64 ±2.56 3.52 ±2.82
CPO group (n�11) 3.54 ±4.89 −4.15 ±2.97
pvalue 0.21 0.04
SpO
2
change
(%)
Control group
(n�11) 0.1 ±0.29 −0.18 ±0.51
CPO group (n�11) −0.36 ±0.60 1.05 ±0.47
pvalue 0.49 0.09
e calculation method of the changes of SBP, DBP, HR, and SpO
2
is: the
change (%) �(post – pre)/pre ×100%. e value represents means ±SEM.
Training: n-back training session. Test: n-back and mental arithmetic tasks
test session.
4Evidence-Based Complementary and Alternative Medicine
CPO group was significantly lower than those of the control
group (p<0.05). ese results indicated that CPO odor
relieved the stress of participants during the test session.
e calculation method of the changes of SBP, DBP,
HR, and SpO
2
is: the change (%) �(post –pre)/pre ×100%.
e value represents means ±SEM. Training: n-back
training session. Test: n-back and mental arithmetic tasks
test session.
3.4. Effects of CPO Intervention on EEG Power Spectrum
Pattern. e EEG data of four 5-min stages of the experi-
ment were analyzed (Figures 3 and 4). ere was a significant
decrease of beta (12.5–30 Hz) power (p<0.05) in the F3
region of the CPO group during the first 5-min odor in-
tervention stage compared with the posttraining stage (after
the n-back training and before the odor intervention;
Figure 3(c)). A decreased trend of beta (12.5–30 Hz) power
could also be observed in the F4 region during the CPO
intervention but showed no significant difference compared
with it in the posttraining stage (p>0.05). ese changes
were not observed in the control treatment group who re-
ceived odorless jojoba oil treatments (Figures 3(a)–3(d)). In
the first 5-min odor intervention stage, the alpha
(7.5–12.5Hz) power in F3, F4, C3, and C4 regions showed a
trend of decrease, but there was no statistical significance
(Figures 3(a) and 3(b) and Figures 4(a) and 4(b)). ere were
no significant changes of beta (12.5–30 Hz) power (p>0.05)
in C3 and C4 regions during each task stage (Figures 4(c)
and 4(d)).
3.5. Evaluation of the Perception of the Environmental Odor.
e two groups of participants showed differences in the
perception of odors in the environment. e score of odor
comfort of the CPO group was higher than that of the
control group, but there was no significant difference
(p>0.05). In terms of the odor intensity, the participants
could clearly sense a stronger aroma of the CPO. e
intensity score of the CPO group was significantly higher
(p<0.05) than that of the control group (Figure 5).
4. Discussion
In this study, n-back and mental arithmetic tasks were used
as stress stimulations, and the effect of CPO odors on
anxiety-relieving was evaluated. e participants first ex-
perienced the training phase of the n-back task and then
received a continuous test phase of the n-back and mental
arithmetic task. Before and after these two stages, the psy-
chological and physiological indexes of the participants were
collected.
e CPO odor showed some significant effects on al-
leviating anxiety that were shown in several indexes in
subjective, physiological, and EEG measurements. e STAI
scale had been used in several previous studies to evaluate
the effect of essential oil interventions on anxiety relief.
Acute inhalation of essential oils with floral, fruity, or herb
smell, such as pelargonium [28], bergamot [29], and salvia
[30] oils, was proved to help reduce the STAI score. In the
present study, the STAI score of the control and CPO group
showed no difference after the n-back training. e STAI
score of the CPO group significantly decreased after the
mental arithmetic test, while the control group did not have
the change. ese results indicated that the woody odor of
CPO might help relieve the anxiety of the participants
compared to an odorless treatment. S-AI and T-AI, which
are two subscales of STAI, represent state anxiety and trait
anxiety, respectively [31]. e S-AI score only decreased
after the mental arithmetic test in the CPO group. Moreover,
the S-AI score of the CPO group was significantly lower than
that of the control group. ese results indicated that the
CPO odors can alleviate the state anxiety of the participants
during the experiment.
e changes in HR and BP are related to people’s ten-
sion. e sympathetic response of the autonomous system
leads to an increase in BP and HR. In some research, these
physiological markers had negative relationships with blood
oxygen saturation [32]. Some previous studies have reported
that smelling or massaging essential oils such as ylang-ylang,
bitter orange, and lavender could reduce HR [33], and a
pleasant smell could bring a decrease in HR [34]. e BP
change was observed after a few essential oil interventions
such as ylang-ylang and bitter orange oil that were thought
to be sedative [19, 20]. ere was no significant difference in
the change of HR, BP (both diastolic and systolic blood
pressure), and blood oxygen saturation between the two
groups before and after the n-back training stage without
odor intervention. After the odor intervention, the HR
changes of participants in the CPO group were significantly
different from those in the control group during the n-back
and mental arithmetic task stage. e HR of the control
group showed an upward trend in the test stage, while that of
the CPO group showed a downward trend. ese results
showed that the inhalation of CPO could alleviate partici-
pants’ tension. Many essential oils containing β-car-
yophyllene, such as Baccharis uncinella and Ocimum
basilicum oils showed some sedative effects [35]. e
#
*
s3s2s1
control
s3s2s1
CPO
0
200
400
600
800
1000
cortisol (ng/ml)
Figure 2: e change of salivary cortisol in control and CPO
groups during three sessions of the experiment. s1: before the
training of the n-back task; s2: after the training of the n-back task;
and s3: after the n-back and mental arithmetic task. Values rep-
resent the mean ±SEM. ∗p<0.05, paired two-tailed Student’s t-test
was used. #p<0.05, unpaired two-tailed Student’s t-test was used.
Evidence-Based Complementary and Alternative Medicine 5
decrease of HR might be related to the inhalation of this
ingredient in the oil. e CPO odor did not affect the BP and
blood oxygen saturation of the participants. is was similar
to a previous study that rosemary and lavender oil could help
relieve anxiety and reduce radial pulse but did not reduce the
BP of participants during a math test [36]. In another study,
significant changes in BP were observed in the lavender oil
group postoperatively [37]. is inconsistency of results
might be due to different experimental tasks design.
Cortisol is a salivary endocrinological stress marker [16].
e hypothalamic-pituitary-adrenal axis (HPA) is one of the
main components of the stress system. Activation of hy-
pothalamic-pituitary-adrenal axis is associated with the
secretion of cortisol [38]. Saliva collection is much more
convenient than blood collection, so it has been used in
many clinical studies. Several previous studies showed that
some essential oils could affect the cortisol contents in saliva
[15, 39]. In the present study, the content of salivary cortisol
of the control group increased in the n-back and mental
arithmetic test stage, while the salivary cortisol of the CPO
group decreased. e salivary cortisol of the CPO group was
also significantly lower than that of the control group. ese
results were consistent with the data presented in HR and
STAI self-report, which indicated that the CPO intervention
could alleviate the anxiety of the participants.
In general, the beta wave activity decreases during the
drowsiness state and increases with a high alertness level
[40]. Many studies have demonstrated that essential oils
affect the EEG spectrum power in rats and humans. In a
previous study, inhalation of essential oil of chamomile,
which was thought to be anxiolytic, led to a decrease of
frontal beta wave activities in females [41]. In the present
study, a remarkable decrease of beta wave power was ob-
served in the left midfrontal region (F3) when the participant
received the CPO intervention. ese results indicated that
inhalation of CPO odor could help relax and relieve anxiety,
which was consistent with the results of STAI, HR, and
cortisol. A decreased trend of alpha wave power was shown
in the F3, F4, C3, and C4 regions of the CPO group after
receiving the odor intervention. ese results were
F3
intervention
post task
post traning
pre training
intervention
post task
post traning
pre training
0
5
10
15
20
25
α % (7.5-12.5 Hz)
control
CPO
(a)
F4
intervention
post task
post traning
pre training
0
5
10
15
20
25
α % (7.5-12.5 Hz)
intervention
post task
post traning
pre training
control
CPO
(b)
F3
*
intervention
post task
post traning
pre training
intervention
post task
post traning
pre training
0
20
40
60
β % (12.5-30 Hz)
control
CPO
(c)
F4
intervention
post task
post traning
pre training
intervention
post task
post traning
pre training
0
20
40
60
β % (12.5-30 Hz)
control
CPO
(d)
Figure 3: e frontal alpha (7.5–12.5Hz) and beta (12.5–30 Hz) activities (% of total 1–30Hz spectrum power) in F3 (a) and (c) and F4 (b)
and (d) during four 5-min stages of the experiment. Pretraining: before the n-back training task; posttraining: after the n-back training task;
intervention: immediately after receiving CPO or control treatment; and posttask: after the mental arithmetic test task. Values represent the
mean ±SEM. ∗p<0.05. Paired two-tailed Student’s t-test was used.
6Evidence-Based Complementary and Alternative Medicine
consistent with a previous study in which the subjects who
received rosemary intervention and obtained lower anxiety
scores exhibited decreasing alpha and beta power [18].
However, this study has potential limitations. In one of
our unpublished studies on odor preference, there was no
significant gender difference in the preference of CPO.
C3
pre training
intervention
post task
post traning
pre training
intervention
post task
post traning
0
5
10
15
20
25
α % (7.5-12.5 Hz)
control
CPO
(a)
C4
pre training
intervention
post task
post traning
pre training
intervention
post task
post traning
0
5
10
15
20
α % (7.5-12.5 Hz)
control
CPO
(b)
C3
0
20
40
60
β % (12.5-30 Hz)
pre training
intervention
post task
post traning
pre training
intervention
post task
post traning
control
CPO
(c)
C4
pre training
intervention
post task
post traning
pre training
intervention
post task
post traning
0
20
40
60
β % (12.5-30 Hz)
control
CPO
(d)
Figure 4: e frontal alpha (7.5–12.5Hz) and beta (12.5–30Hz) activities (% of total 1–30 Hz spectrum power) in C3 (a) and (c) and C4
(b) and (d) during four 5-min stages of the experiment. Pretraining: before the n-back training task; posttraining: after the n-back training
task; intervention: immediately after receiving CPO or control treatment; and posttask: after the mental arithmetic test task. Values
represent the mean ±SEM. Paired two-tailed Student’s t-test was used.
CPO
control
Comfort
-3 -2 -1-4 1 2 3 4 5 706
score
(a)
CPO
control
Intensity
12345670
score
*
(b)
Figure 5: e subjective evaluation of the comfort and intensity of the odors. e median line of the histogram represents the average of the
value. ∗p<0.05. Unpaired two-tailed Student’s t-test was used.
Evidence-Based Complementary and Alternative Medicine 7
However, the sample size of the present study was small, and
there were more females than males. Whether there were
gender differences in the anxiety-relieving function of CPO
could not be discussed in this study. Furthermore, in the
process of odor intervention, the essential oil molecules in
the experimental environment space were accumulated due
to the continuous running of the aroma diffuser. e odor
concentration was not strictly controlled. is problem is
common in many clinical studies of aromatherapy [42]. A
large sample size and an environment with controlled odor
concentration will be helpful to explore if the anxiolytic
functions vary at different concentrations in further study.
5. Conclusion
CPO with a woody odor is popular in aromatherapy these
years and is recommended for anxiety-relieving in aroma-
therapy practice. In the present study, a randomized con-
trolled trial was designed to evaluate the anxiety-relieving
effect of CPO inhalation in 22 adults. N-back and mental
arithmetic tasks were used as stress stimulation. e results
showed that the CPO odor could well alleviate the anxiety of
participants compared to the control environment. e S-AI
score of the CPO treated group decreased after the n-back
and mental arithmetic test, and it was significantly lower
than that of the control group. e HR and salivary cortisol
of participants who received CPO intervention significantly
decreased during the n-back and mental arithmetic tests.
Furthermore, a remarkable decrease of beta wave activity
was observed in the left midfrontal region (F3) when the
participant received the CPO intervention. Studies using a
larger sample size and environments with controllable odor
concentrations will be helpful to further explore the anxi-
olytic function of CPO.
Data Availability
Data generated or analyzed during this study are included in
this article. Further enquiries are available on reasonable
request to the corresponding author.
Additional Points
Highlights. (i) A randomized controlled trial indicated that
inhalation of copaiba oil could help relieve the anxiety of the
participant during mental workload. (ii) e STAI scores
and physiological indexes related to anxiety including heart
rate and salivary cortisol decreased after the participant
received the copaiba oil inhalation treatment. (iii) A decrease
in left midfrontal beta wave activities of EEG was observed
during the copaiba oil treatment.
Conflicts of Interest
e authors declare that there are no conflicts of interest.
Authors’ Contributions
Nan Zhang contributed to methodology, did the investi-
gation and formal analysis, and wrote the paper. Jie Chen did
the investigation and formal analysis. Lei Yao conceptual-
ized the study, supervised, and contributed to funding ac-
quisition. Yan Wen Dong contributed to acquiring
resources.
Acknowledgments
e authors deeply thank volunteers for their participation
and cooperation. is work was supported by the National
Key Research and Development Program of China (grant
number: 2019YFA0706200).
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